US10772974B2 - Compositions and methods for cardiac regeneration - Google Patents

Compositions and methods for cardiac regeneration Download PDF

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US10772974B2
US10772974B2 US15/037,195 US201415037195A US10772974B2 US 10772974 B2 US10772974 B2 US 10772974B2 US 201415037195 A US201415037195 A US 201415037195A US 10772974 B2 US10772974 B2 US 10772974B2
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Anthony Rosenzweig
Vassilios J. Bezzerides
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Boston Childrens Hospital
Beth Israel Deaconess Medical Center Inc
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Definitions

  • This invention was made with government support under HL110733, HL114352, HL007572, HL073734, GM007226 and TR000901 awarded by NIH. The government has certain rights in the invention.
  • Heart disease and its manifestations including coronary artery disease, myocardial infarction, congestive heart failure and cardiac hypertrophy, clearly presents a major health risk in the United States today.
  • the cost to diagnose, treat and support patients suffering from these diseases is well into the billions of dollars.
  • a particularly severe manifestation of heart disease is myocardial infarction.
  • Myocardial infarction (MI) more commonly known as a heart attack, is a medical condition that occurs when the blood supply to a part of the heart is interrupted, most commonly due to rupture of a vulnerable plaque.
  • the resulting ischemia or oxygen shortage causes damage and potential death of heart tissue. It is the leading cause of death for both men and women throughout the world.
  • coronary heart disease is responsible for 1 in 5 deaths, and some 7,200,000 men and 6,000,000 women are living with some form of coronary heart disease. Of these, 1,200,000 people suffer a new or recurrent coronary attack every year, and about 40% of them die as a result of the attack. This means that roughly every 65 seconds, an American dies of a coronary event.
  • MI events are either “silent” or are clinically unrecognized, but are nonetheless encompassed within this definition.
  • the appearance of cardiac markers in the circulation generally indicates myocardial necrosis and is a useful adjunct to diagnosis.
  • Such markers included ST-elevation MI (STEMI), non-ST-elevation MI (NSTEMI), and unstable angina.
  • the difference in conduction velocity between injured and uninjured tissue can trigger re-entry or a feedback loop that is believed to be the cause of many lethal arrhythmias. Cardiac output and blood pressure may fall to dangerous levels, which can lead to further coronary ischemia and extension of the infarct.
  • MicroRNAs are small, non-protein coding RNAs of about 18 to about 25 nucleotides in length that are derived from individual miRNA genes, from introns of protein coding genes, or from poly-cistronic transcripts that often encode multiple, closely related miRNAs. See review by Carrington et al. (Science 301(5631):336-338, 2003). MiRNAs are thought to act as repressors of target mRNAs by promoting their degradation, when their sequences are perfectly complementary, or by inhibiting translation, when their sequences contain mismatches.
  • MiRNAs are transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III; see Qi et al., Cellular & Molecular Immunology 3:411-419, 2006) and arise from initial transcripts, termed primary miRNA transcripts (pri-miRNAs), that are generally several thousand bases long.
  • Pri-miRNAs are processed in the nucleus by the RNase Drosha into about 70- to about 100-nucleotide hairpin-shaped precursors (pre-miRNAs). Following transport to the cytoplasm, the hairpin pre-miRNA is further processed by Dicer to produce a double-stranded miRNA.
  • RISC RNA-induced silencing complex
  • One aspect of the invention provides a method of promoting physiological cardiomyocyte growth or proliferation in vivo, the method comprising promoting, in an adult subject in need thereof, expression or activity of a CITED4 (CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4) polypeptide or a functional fragment or fusion protein thereof from an expression construct.
  • CITED4 CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4
  • the invention provides a method of treating a heart disease treatable by cardiomyocyte regeneration and/or proliferation, the method comprising promoting, in an adult subject in need thereof, expression or activity of a CITED4 (CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4) polypeptide or a functional fragment or fusion protein thereof from an expression construct.
  • CITED4 CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4
  • the invention provides a method of promoting physiological cardiomyocyte growth or proliferation, the method comprising increasing the level of microRNA-222 (miR-222) or a precursor (e.g., pre-miR-222) or a mimic thereof in a cardiomyocyte or precursor thereof.
  • the invention provides a method of treating a heart disease treatable by cardiomyocyte regeneration and/or proliferation, the method comprising increasing, in an adult subject in need thereof, the level of miR-222 or a precursor (e.g., pre-miR-222) or a mimic thereof.
  • the expression or activity of the CITED4 polypeptide, or the level of miR-222 is increased in a cardiomyocyte of the subject.
  • the expression or activity of the CITED4 polypeptide, or the level of miR-222 is increased by administering to the subject an expression construct encoding the CITED4 polypeptide, or miR-222.
  • the expression construct is administered by intravenous administration; by direct injection into cardiac tissue; or by oral, transdermal, sustained release, controlled release, delayed release, suppository, subcutaneous, intramuscular, catheter or sublingual administration.
  • the expression construct is a viral vector, such as an adenoviral vector or an adeno-associated viral (AAV) vector, the latter of which may be AAV1, AAV2, or AAV9, or a combination thereof.
  • a viral vector such as an adenoviral vector or an adeno-associated viral (AAV) vector, the latter of which may be AAV1, AAV2, or AAV9, or a combination thereof.
  • AAV adeno-associated viral
  • the AAV vector is delivered to the subject via intracoronary infusion.
  • the expression of CITED4 or level of miR-222 is increased in the cardiomyocyte or precursor thereof by contacting the cardiomyocyte or precursor thereof with a synthetic, modified RNA.
  • the heart disease is myocardial infarction or ischemic injury; adverse remodeling after ischemic injury or infarction; myocarditis; heart failure (congestive), cardiomyopathy (e.g., ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy, tachycardia-mediated cardiomyopathy, stress-induced (takotsubo) cardiomyopathy, amyloid cardiomyopathy, arrhythmogenic right ventricular dysplasia, or unclassified cardiomyopathy, left ventricular noncompaction or endocardial fibroelastosis); valvular heart disease (e.g., aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, mitral prolapse, pulmonary stenosis, pulmonary regurgitation, tricuspid stenosis, or tricuspid regurgitation).
  • cardiomyopathy e.g.,
  • therapeutic efficacy is achieved by alleviating at least one symptom of the heart disease (e.g., heart failure), or by inhibiting or retarding the worsening of the symptom.
  • therapeutic efficacy is measured by a decrease in a symptom of heart failure (e.g., as measured by New York Heart Association class, Minnesota Living With Heart Failure Questionnaire), an augment in functional status (e.g., as measured by 6-minute walk test, peak maximum oxygen consumption), a decrease in natriuretic peptide level (e.g., N-terminal prohormone brain natriuretic peptide), and/or beneficial reverse left ventricular (LV) remodeling (left ventricular ejection fraction, left ventricular end-systolic volume).
  • a symptom of heart failure e.g., as measured by New York Heart Association class, Minnesota Living With Heart Failure Questionnaire
  • an augment in functional status e.g., as measured by 6-minute walk test, peak maximum oxygen consumption
  • natriuretic peptide level e.g.
  • the method further comprises administering to the subject a second cardiac therapy.
  • the second therapy may be selected from the group consisting of a ⁇ blocker, an ionotrope, a diuretic, ACE-I, All antagonist, BNP, a Ca 2+ -blocker, an endothelin receptor antagonist, and an HDAC inhibitor.
  • the heart disease is myocardial infarction, and wherein fibrosis and/or apoptosis in the infarct zone is reduced.
  • the subject is a human.
  • Another aspect of the invention provides an expression construct capable of directing in vivo expression of a CITED4 polypeptide or a functional fragment or fusion protein thereof.
  • Another aspect of the invention provides an expression construct capable of directing expression of miR-222 or a precursor or a mimic thereof.
  • Another aspect of the invention provides a method of identifying miR-222 mimics, the method comprising: (1) contacting a first cardiomyocyte with miR-222, or an expression vector that expresses miR-222 in the first cardiomyocyte, and determining a first extent of cardiomyocyte growth or proliferation; (2) contacting a second cardiomyocyte with a candidate compound under substantially the same condition as in (1), and determining a second extent of cardiomyocyte growth or proliferation; (3) comparing the first extent of cardiomyocyte growth or proliferation with the second extent of cardiomyocyte growth or proliferation; wherein the candidate compound is identified as a miR-222 mimic when the first extent of cardiomyocyte growth or proliferation is substantially the same as the second extent of cardiomyocyte growth or proliferation.
  • compositions and kits of the invention can be used to achieve methods of the invention.
  • the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Alternatively, “about” is within 5% of the value being modified.
  • FIG. 1 shows that CITED4 induces proliferation of neonatal cardiomyocytes in vitro.
  • rat neonatal cardiomyocytes were treated with either an adenovirus overexpressing CITED4 or an siRNA directed against CITED4. Cells were then stained against ⁇ -actinin and ki67, and positive cells were counted in 20 ⁇ view fields from 15 random images per group.
  • rat neonatal cardiomyocytes were treated with control siRNA, C/EBPb siRNA+control siRNA, or C/EBPb siRNA+Cited4 siRNA, followed by assay of BrdU incorporation. Data is pooled from three experiments and presented as percent of control.
  • FIG. 2 shows that CITED4 expression induces markers of (adult) cardiomyocyte proliferation in vivo, including the cardiac marker Troponin T (cTnT) and EdU (a thymidine analogue that is incorporated into newly formed DNA). No increase in EdU incorporation was seen in non-cardiomyocyte lineages, including endothelial and fibroblast cells.
  • cTnT cardiac marker Troponin T
  • EdU a thymidine analogue that is incorporated into newly formed DNA
  • FIG. 3 shows that CITED4 expression promotes recovery and repair after ischemic injury in an animal model.
  • FIGS. 4A-4F show that miR-222 is necessary for exercise-induced cardiac growth in vivo.
  • sedentary or swum mice were intravenously injected with LNA-antimiR-222 or control LNA-antimiR for 3 weeks prior to quantification of cardiac miR-222 to demonstrate effective reduction after injection of LNA-antimiR-222.
  • FIGS. 4A-4F show that miR-222 is necessary for exercise-induced cardiac growth in vivo.
  • HW/BW heart weight/body weight
  • HW/TL heart weight/tibia length
  • FIG. 5 shows that treatment of exercising animals with LNA-anti-miR-222 blocks the induction of phospho-histone-H3, a marker of cell proliferation, specifically in the cardiomyocyte lineage.
  • FIG. 6 shows that miR-222 expression promotes functional recovery and reduces scar formation after ischemic injury.
  • FIG. 7 shows that direct targets of miR-222 in cardiomyocytes include p27, HIPK1 and 2, and Homeobox-1, as shown in expression studies and 3′-UTR luciferase assays with wild-type and mutant 3′-UTRs.
  • FIG. 8 suggests distinct effects of miR-222 targets in vitro.
  • siRNA knockdown of Homeobox-1 is sufficient to induce cardiomyocytes growth in size (but not proliferation), while knockdown of p27 and HIPK1 are sufficient to drive proliferation of cardiomyocytes in vitro (but not size increase).
  • FIGS. 9A-9E show differentially expressed microRNAs in hearts from exercised mice.
  • FIGS. 10A-10C show functional effects of validated microRNAs in neonate cardiomyocytes.
  • FIG. 10A shows immunohistochemistry against sarcomeric ⁇ -actinin followed by quantification of cell area in neonatal cardiomyocytes transfected with the indicated microRNAs or treated with phenylephrine (PE).
  • FIG. 10B shows flow cytometry analysis of EdU incorporation in neonatal cardiomyocytes transfected with control precursor, precursors of the indicated miRNAs, or precursor for has-miR-590-3p as a positive control. Cumulative data from three independent experiments are shown.
  • FIG. 10C shows qRT-PCR analysis of the relative ratio of ⁇ / ⁇ -MHC in neonate cardiomyocytes transfected with indicated miR precursors. Cumulative data from three independent experiments are shown. All data are represented as mean ⁇ SEM. *p ⁇ 0.05 versus respective control using Student's t-test.
  • FIGS. 11A-11E show that miR-222 is necessary and sufficient for cardiomyocyte hypertrophy and proliferation.
  • FIGS. 11A and 11B show immunohistochemical staining for sarcomeric ⁇ -actinin (image not shown) followed by quantification of cardiomyocyte area as described in methods. Cells were transfected with control or miR-222 precursor in FIG. 11A and with control antimiR (ctl-anti) or antimiR-222 (anti-222) in FIG. 11B . At least 200 cells were quantified in each group. These data demonstrate that miR-222 is necessary and sufficient to induce cardiomyocyte hypertrophy in vitro.
  • FIG. 11A and 11B show immunohistochemical staining for sarcomeric ⁇ -actinin (image not shown) followed by quantification of cardiomyocyte area as described in methods. Cells were transfected with control or miR-222 precursor in FIG. 11A and with control antimiR (ctl-anti) or antimiR-222 (anti-222)
  • FIG. 11C shows quantification of EdU incorporation staining (left panel), Ki67 staining (middle panel), and cell number (right panel) from primary NRVMs transfected with control precursor (ctl-pre) or miR-222 precursor (pre-222).
  • FIG. 11D shows quantification of EdU incorporation staining, Ki67 staining, and cell number from NRVMs transfected with control antimiR (ctl-anti) or antimiR-222 (anti-222). These data demonstrate miR-222 is necessary and sufficient to induce neonatal cardiomyocyte proliferation in vitro.
  • FIG. 11E shows qRT-PCR analysis of cardiomyocyte gene expression in NRVMs treated with control (ctl-pre) or miR-222 precursor (pre-222).
  • FIG. 12A-12B show baseline characteristics of Tg-miR-222 mice.
  • FIG. 12A shows qRT-PCR analysis of microRNA expression in hearts of tTA single (miR-222 ⁇ /tTA + ) and double (miR-222 + /tTA + ) 3 month old transgenic mice 4 weeks after doxycycline removal from chow to induce miR-222 expression in double-transgenic mice. Data are shown as fold induction of microRNA expression normalized to U6. Cumulative data from 4 to 6 mice for each genotype are shown.
  • FIG. 12A shows qRT-PCR analysis of microRNA expression in hearts of tTA single (miR-222 ⁇ /tTA + ) and double (miR-222 + /tTA + ) 3 month old transgenic mice 4 weeks after doxycycline removal from chow to induce miR-222 expression in double-transgenic mice. Data are shown as fold induction of microRNA expression normalized to U6. Cumulative data from 4 to 6
  • FIGS. 13A-13D show that cardiac-specific expression of miR-222 protects against adverse remodeling and dysfunction after ischemic injury.
  • FIG. 13A demonstrates that there is no difference in initial infarct size or the area-at-risk (AAR), in miR-222 expressing hearts.
  • Tiphenyltetrazolium chloride (TTC) staining was used to delineate infarct area, and fluorescent microsphere distribution was used to define the AAR in hearts from tTA single (miR-222 ⁇ /tTA + ) and double transgenic (miR-222 + /tTA + ) mice, 24 hours after reperfusion after 30 minutes of ischemia.
  • TTC area-at-risk
  • LVIDs cardiac fractional shortening and left ventricular internal dimension in systole
  • FIG. 14A-14F show miR-222 targets in cardiomyocytes.
  • qRT-PCR and immunoblotting were used to analyze RNA and protein levels of the four putative miR-222 targets in neonatal cardiomyocytes treated with control precursor (ctl-pre), miR-222 precursor (pre-222), control antimiR (ctl-anti), or antimiR-222 (anti-222), as indicated.
  • Data are shown as fold induction of gene expression normalized to U6 in ( FIG. 14A ). These data demonstrate that miR-222 decreases RNA and protein levels for all four targets in primary cardiomyocytes.
  • HSP90 was used as a loading control.
  • FIG. 14D flow cytometry for EdU incorporation in neonatal cardiomyocytes transfected with the indicated siRNAs demonstrates that knockdown of p27 or HIPK1 increases EdU incorporation in cardiomyocytes.
  • FIGS. 14E and 14F neonatal cardiomyocytes were stained for sarcomeric ⁇ -actinin before quantification of cardiomyocyte number and area. Knockdown of p27 or HIPK1 increases cardiomyocyte proliferation ( FIG. 14E ) while Hmbox1 knockdown increases cardiomyocyte size ( FIG. 14F ). At least 200 cells in 30 images were quantified in each group. Data represent the mean ⁇ SEM from at least three independent experiments. Scale bar: 100 ⁇ m. *p ⁇ 0.05, **p ⁇ 0.01 versus respective control using Student's test.
  • FIG. 15 shows that plasma miR-222 levels in heart failure patients are increased after acute exercise.
  • the figure shows qRT-PCR analysis of miR-222 levels in plasma from heart failure patients before (pre-exercise) and after (post-exercise) acute cardiopulmonary exercise using a bicycle ergometer. miR-222 expression are shown as mean ⁇ standard error from 28 patients, normalized to exogenously added cel-miR-39. *p ⁇ 0.01 using Student's test.
  • FIG. 16 shows tissue-specific (e.g., heart-specific) expression of miR-222 in vivo using AAV9 vector.
  • the present invention is based, at least in part, on the surprising discovery that, although forced expression of a CITED4 polypeptide fails to stimulate adult cardiomyocyte (CM) proliferation in vitro, it is nevertheless sufficient to stimulate CM growth and proliferation in vivo.
  • CM cardiomyocyte
  • the present invention is also based, at least in part, on the discovery that microRNA-222 (miR-222) also promotes cardiomyocyte (CM) proliferation, and that the full beneficial effect of miR-222 may not be fully recapitulated by targeting individual members of a multitude of miR-222 downstream target genes.
  • miR-222 also promotes cardiomyocyte (CM) proliferation
  • the discoveries provide new and useful methods and reagents for treating or preventing a host of cardiovascular diseases and pathological conditions of the heart, including myocardial infarction, heart failure, and scar formation (fibrosis) resulting therefrom, in animals such as in humans, by increasing the expression, activity, or level of a CITED4 polypeptide (or functional fragments or fusions thereof), and/or miR-222 (or precursors or mimics thereof).
  • the invention provides a method of promoting physiological cardiomyocyte growth (increase in cell size but not cell proliferation) or proliferation (increase in cell number) in vivo, the method comprising promoting, in an adult subject in need thereof, expression or activity of a CITED4 (CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4) polypeptide or a functional fragment or fusion protein thereof.
  • CITED4 CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4
  • the invention provides a method of treating a heart disease treatable by cardiomyocyte regeneration and/or proliferation, the method comprising promoting, in an adult subject in need thereof, expression or activity of a CITED4 polypeptide or a functional fragment or fusion protein thereof.
  • heart failure is broadly used to mean any condition in which the function of the heart is inadequate to meet the systemic needs of the metabolic, whether the impaired function is due to reduced contraction (systolic dysfunction) or due to reduce relaxation (diastolic dysfunction). In both conditions, diastolic pressures in the heart increase, resulting in congestion and edema in the tissues. Most frequently, heart failure is caused by decreased contractility of the myocardium, resulting from MI or reduced coronary blood flow; however, many other factors may result in heart failure, including damage to the heart valves, vitamin deficiency, and primary cardiac muscle disease.
  • heart failure is now recognized to result from impaired relaxation of the heart, whether due to scarring (fibrosis), abnormal calcium handling and/or thickening of the heart muscle (hypertrophy).
  • scarring fibrosis
  • calcium handling and/or thickening of the heart muscle hypertrophy.
  • heart failure is generally believed to involve disorders in several cardiac autonomic properties, including sympathetic, parasympathetic, and baroreceptor responses.
  • heart failure is used broadly to encompass all of the sequelae associated with heart failure, such as shortness of breath, pitting edema, an enlarged tender liver, engorged neck veins, pulmonary rales, decreased energy and fatigue, and the like including laboratory findings associated with heart failure.
  • treatment encompasses the amelioration, cure, maintenance (i.e., the prevention of relapse), improvement, and/or reversal of the symptoms of a cardiovascular disease or pathological condition being treated.
  • Treatment after a disease or disorder has started or manifested aims to reduce, ameliorate, or altogether eliminate the disorder, and/or its associated symptoms, to prevent it from becoming worse, or to prevent the disorder from re-occurring once it has been initially eliminated (i.e., to prevent a relapse).
  • treatment does not include prevention.
  • treatment may include the improvement and/or reversal of the diminished ability of the heart to pump blood or its impaired relaxation.
  • Improvement in the physiologic function of the heart may be assessed using any of the measurements described herein (e.g., measurement of ejection fraction, fractional shortening, left ventricular internal dimension, heart rate, etc.), as well as any effect upon the animal's survival.
  • the method is not subjecting the subject to physical exercise. In certain embodiments, the method comprises promoting expression or activity of the CITED4 polypeptide or a functional fragment or fusion protein thereof from an expression construct, preferably in heart cells or cardiomyocytes of the subject.
  • the invention provides a method of promoting physiological cardiomyocyte growth or proliferation, the method comprising increasing the level of microRNA-222 (miR-222) or a precursor (e.g., pre-miR-222) or a mimic thereof in a cardiomyocyte or precursor thereof.
  • the invention provides a method of treating a heart disease treatable by cardiomyocyte regeneration and/or proliferation, the method comprising increasing, in an adult subject in need thereof, the level of miR-222 or a precursor (e.g., pre-miR-222) or a mimic thereof.
  • the method is not subjecting the subject to physical exercise. In certain embodiments, the method comprises increasing the level of miR-222 or a precursor (e.g., pre-miR-222) or a mimic thereof, preferably in heart cells or cardiomyocytes of the subject.
  • a precursor e.g., pre-miR-222
  • a mimic thereof preferably in heart cells or cardiomyocytes of the subject.
  • the term “subject” include any vertebrate species, particularly mammals, including, without limitation, humans and other non-human primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like).
  • the subject is a mammal.
  • the subject is a human.
  • the CITED4 polypeptide and/or miR-222 used in the methods of the invention are from the same species of the subject.
  • the CITED4 polypeptide or miR-222 to be used in the methods of the invention are of human origin.
  • the CITED4 polypeptide and/or miR-222 used in the methods of the invention may be from a different species, such as a closely related species, or a heterologous sequence sharing at least about 80, 85, 90, 95% sequence identity.
  • the expression or activity of the CITED4 polypeptide, or the level of miR-222 is increased by administering to the subject an expression construct encoding the CITED4 polypeptide, or miR-222.
  • an expression construct encoding the CITED4 polypeptide, or miR-222.
  • the expression construct may be administered by intravenous administration; by direct injection into cardiac tissue; or by oral, transdermal, sustained release, controlled release, delayed release, suppository, subcutaneous, intramuscular, catheter or sublingual administration.
  • the expression construct may be a viral vector, such as an adenoviral vector or an adeno-associated viral (AAV) vector.
  • AAV vectors include AAV1, AAV2, and AAV9.
  • the AAV1 vector may be delivered to the subject via intracoronary infusion.
  • Jessup et al. ( Circulation 124: 304-313, 2011) reported that an Adeno-Associated Virus type 1 (AAV1) viral vector was used to exogenously express sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) in patients with advanced heart failure, demonstrating safety and feasibility of the gene transfer therapy, as well as therapeutic efficacy.
  • AAV1 viral vector was used to exogenously express sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) in patients with advanced heart failure, demonstrating safety and feasibility of the gene transfer therapy, as well as therapeutic efficacy.
  • SERCA2a sarcoplasmic reticulum Ca 2+ -ATPase
  • the expression of CITED4 or level of miR-222 is increased in the cardiomyocyte or precursor thereof by contacting the cardiomyocyte or precursor thereof with a synthetic, modified RNA.
  • the heart disease is myocardial infarction or ischemic injury; adverse remodeling after ischemic injury or infarction; myocarditis; heart failure (congestive), cardiomyopathy (e.g., ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy, tachycardia-mediated cardiomyopathy, stress-induced (takotsubo) cardiomyopathy, amyloid cardiomyopathy, arrhythmogenic right ventricular dysplasia, or unclassified cardiomyopathy, left ventricular noncompaction or endocardial fibroelastosis); valvular heart disease (e.g., aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, mitral prolapse, pulmonary stenosis, pulmonary regurgitation, tricuspid stenosis, or tricuspid regurgitation).
  • cardiomyopathy e.g.,
  • therapeutic efficacy is achieved based on the methods of the invention, by alleviating at least one symptom of the heart disease (e.g., heart failure), or by inhibiting or retarding the worsening of the symptom.
  • the heart disease e.g., heart failure
  • therapeutic efficacy may be measured by a decrease in a symptom of heart failure (e.g., as measured by New York Heart Association class, Minnesota Living With Heart Failure Questionnaire), an augment in functional status (e.g., as measured by 6-minute walk test, peak maximum oxygen consumption), a decrease in natriuretic peptide level (e.g., N-terminal prohormone brain natriuretic peptide), and/or beneficial reverse left ventricular (LV) remodeling (left ventricular ejection fraction, left ventricular end-systolic volume).
  • a symptom of heart failure e.g., as measured by New York Heart Association class, Minnesota Living With Heart Failure Questionnaire
  • an augment in functional status e.g., as measured by 6-minute walk test, peak maximum oxygen consumption
  • natriuretic peptide level e.g., N-terminal prohormone brain natriuretic peptide
  • beneficial reverse left ventricular (LV) remodeling left ventricular ejection fraction, left ventricular end-sy
  • the methods of the invention further comprise administering to the subject a second cardiac therapy, such as one selected from the group consisting of: a ⁇ blocker, an ionotrope, a diuretic, ACE-I, All antagonist, BNP, a Ca 2+ -blocker, an endothelin receptor antagonist, and an HDAC inhibitor.
  • a second cardiac therapy such as one selected from the group consisting of: a ⁇ blocker, an ionotrope, a diuretic, ACE-I, All antagonist, BNP, a Ca 2+ -blocker, an endothelin receptor antagonist, and an HDAC inhibitor.
  • the heart disease is myocardial infarction, and wherein fibrosis and/or apoptosis in the infarct zone is reduced.
  • Another aspect of the invention provides an expression construct capable of directing in vivo expression of a CITED4 polypeptide or a functional fragment or fusion protein thereof.
  • Still another aspect of the invention provides an expression construct capable of directing expression of miR-222 or a precursor (e.g., pre-miR-222) or a mimic thereof.
  • compositions comprising a subject CITED4 polypeptide or a functional fragment or fusion protein thereof, or miR-222 or a precursor (e.g., pre-miR-222) or mimic thereof, or an expression construct encoding the same, and a pharmaceutically acceptable carrier, excipient, or buffer.
  • the pharmaceutically acceptable carrier or excipient may be suitable for rendering the compound or mixture administrable orally as a tablet, capsule or pill, or parenterally, intravenously, intradermally, intramuscularly or subcutaneously, or transdermally.
  • the active ingredients may be admixed or compounded with any conventional, pharmaceutically acceptable carrier or excipient.
  • the term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions of this invention, its use in the therapeutic formulation is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical formulations.
  • a composition is said to be a “pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known in the art. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990).
  • Another aspect of the invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Associated with such container(s) can be various written materials (written information) such as instructions (indicia) for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Yet another aspect of the invention provides a method to screen for/identify miR-222 mimics among a plurality of candidates, comprising (1) contacting a first cardiomyocyte with miR-222, or an expression vector that expresses miR-222 in the first cardiomyocyte, and determining a first extent of cardiomyocyte growth or proliferation; (2) contacting a second cardiomyocyte with a candidate compound under substantially the same condition as in (1), and determining a second extent of cardiomyocyte growth or proliferation; (3) comparing the first extent of cardiomyocyte growth or proliferation with the second extent of cardiomyocyte growth or proliferation; wherein the candidate compound is identified as a miR-222 mimic when the first extent of cardiomyocyte growth or proliferation is substantially the same as the second extent of cardiomyocyte growth or proliferation.
  • the first or second extent of cardiomyocyte growth or proliferation is measured by the presence/absence or level of expression of a marker gene, such as a marker gene for cardiomyocyte growth or proliferation.
  • a marker gene such as a marker gene for cardiomyocyte growth or proliferation.
  • Exemplary marker genes include Ki67, EdU, phospho-histone H3 (PPH3), TnT, and/or Aurora B kinase in cardiomyocytes identified by virtue of Troponin T expression.
  • the candidate compound is a chemically modified nucleic acid.
  • the chemically modified nucleic acid may have substantially identical sequence as the wildtype miR-222 sequence or the CITED4 mRNA sequence, but contains one or more modified nucleic acid that enhances serum stability, cellular in-take, and/or nuclease resistance, and/or reduces the host immune or inflammatory response.
  • nucleic acid molecules with modifications may resist degradation by serum ribonucleases, and may have increased potency.
  • modifications base, sugar and/or phosphate
  • may resist degradation by serum ribonucleases and may have increased potency.
  • Eckstein et al. International Publication No. WO 92/07065; Perrault et al, 1990 Nature 344:565; Pieken et al., 1991 , Science 253:314; Usman and Cedergren, 1992 , Trends in Biochem. Sci. 17:334; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2′amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992 , TIBS. 17:34; Usman et al., 1994 , Nucleic Acids Symp. Ser. 31:163; Burgin et al., 1996 , Biochemistry, 35:14090).
  • nuclease resistant groups for example, 2′amino, 2′-C-allyl, 2′-flouro, 2′-O-methyl, 2′-H
  • nucleotide base modifications for a review see Usman and Cedergren, 1992 , TIBS. 17:34; Usman et al., 1994 , Nucleic Acids Symp. Ser. 31:163; Burgin et al.,
  • miR-222 or precursor or mimic thereof contains DNA, RNA, modification thereof (e.g., LNA or PNA etc.), and/or combinations thereof.
  • Locked Nucleic Acids or LNAs comprise sugar-modified nucleotides that resist nuclease activities (thus highly stable), and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., 33(1): 439-447, 2005; Braasch et al., Biochemistry, 42:7967-7975, 2003; Petersen et al., Trends Biotechnol., 21:74-81, 2003). These molecules have 2′-0,4′-C-ethylene-bridged nucleic acids, with possible modifications such as 2′-deoxy-2′′-fluorouridme.
  • LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3′-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10° C. per base.
  • PNAs Peptide Nucleic Acids
  • PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone, which is highly resistant to nuclease digestion, and imparts improved binding specificity to the molecule (Nielsen et al., Science, 254:1497-1500, 2001).
  • the oligonucleotide of the invention comprises Morpholino nucleic acid analog, or “PMO” (phosphorodiamidate morpholino oligo).
  • Morpholinos are synthetic nucleic acid analogs that bind to complementary sequences of RNA by standard nucleic acid base-pairing. Structurally, Morpholinos are similar to DNA in that Morpholinos have standard nucleic acid bases. However, those bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
  • miR-222 or precursor or mimic thereof contains Glycol Nucleic A (GNA), which is a synthesized polymer similar to DNA or RNA but differing in the composition of its backbone.
  • GNA Glycol Nucleic A
  • DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas GNA's backbone is composed of repeating glycol units linked by phosphodiester bonds.
  • the glycol unit has just three carbon atoms, yet still shows Watson-Crick base pairing, and the Watson-Crick base pairing is much more stable in GNA than its natural counterparts DNA and RNA as it requires a high temperature to melt a duplex of GNA.
  • the 2,3-dihydroxypropylnucleoside analogues were first prepared by Ueda et al. (1971).
  • miR-222 or precursor or mimic thereof contains Threose Nucleic Acid (TNA), which is a synthetic nucleic acid analog similar to DNA or RNA but differing in the composition of its backbone.
  • TNA's backbone is composed of repeating threose units linked by phosphodiester bonds.
  • TNA can hybridize with RNA and DNA in a sequence-specific manner.
  • TNA is also capable of Watson-Crick pair bonding, and forming a double helix structure.
  • MicroRNA-222 (miR-222)
  • the invention provides methods of using a subject miR-222 microRNA or precursor or mimic thereof for treating the various cardiovascular diseases or pathological conditions described here.
  • pre-miRNA is the process product of pri-miRNA by Drosha/Pasha, and can be exported from nucleus of the infected cell to the cytoplasm by the RAN-GTPase Exportin 5.
  • the pre-miRNA may be further bound and processed by another RNase III Dicer to produce a double-stranded complex of miR and its complementary sequence, which miR is subsequently loaded into the mi-RISC complex, and forms a complex with an Argonaute protein (e.g., AGO2).
  • an Argonaute protein e.g., AGO2
  • the precursor may also include sequences (natural or synthetic) not identical to the pre-miR-222 or pri-miR-222, but can nevertheless be similarly processed by the same RNase III.
  • the sequence of a human miR-222 precursor is:
  • the sequence of the mature human miR-222 is CUCAGUAGCCAGUGUAGAUCCU (SEQ ID NO: 2; Accession: MIMAT0000279).
  • the miR-222 sequences are highly conserved throughout evolution. BLAST search using the human miR-222 sequence above as a query retrieved numerous related human and non-human miR-222 sequences.
  • the Macaca mulatta mir-222 sequence NR_032459 is 99% (109/110) identical to the human sequence SEQ ID NO: 1 above;
  • the Equus caballus (horse) microRNA mir-222 NR_033080 is 98% (108/110) identical to the human sequence SEQ ID NO: 1 above;
  • the Bos taurus (cattle) microRNA mir-222 NR_030882 is 97% (107/110) identical to the human sequence SEQ ID NO: 1 above;
  • the Rattus norvegicus (rat) microRNA 222 NR_031935 is 96% (90/94) identical to nucleotides 17-110 of the human sequence SEQ ID NO: 1 above;
  • the Mus musculus (mouse) microRNA-222 NR_029807 is 96% (
  • the miR-222 or precursor is a human sequence.
  • the miR-222 or precursor is a mammalian sequence, such as a non-human primate sequence.
  • the subject agent is a miR-222 precursor, such as a pri- or pre-miR-222, or a sequence comprising SEQ ID NO: 1.
  • the subject agent is a miR-222 mimic or pri-miR-222 or pre-miR-222 mimic, such as a modified pri-miR-222 or pre-miR-222 containing one or more modified nucleic acid that enhances serum stability, cellular in-take, and/or nuclease resistance, and/or reduces the host immune or inflammatory response.
  • the invention provides methods of using a subject CITED4 related polypeptide for treating the various cardiovascular diseases or pathological conditions described here.
  • CBP/p300-Interacting Transactivator with Glu/Asp-rich carboxyl terminal Domain, 4
  • CBP/p300-Interacting Transactivator with ED-rich carboxy-terminal Domain 4 are used interchangeably herein to refer to a family of mammalian polypeptides that act as transcriptional coactivator for Transcription Factor AP-2 (TFAP2)/AP-2; that enhance estrogen-dependent transactivation mediated by estrogen receptors; that may function as an inhibitor of transactivation by HIF1A by disrupting HIF1A interaction with CREB Binding Protein (CREBBP); and that may be involved in regulation of gene expression during development and differentiation of blood cells, endothelial cells and mammary epithelial cells.
  • TFAP2 Transcription Factor AP-2
  • CREBBP CREB Binding Protein
  • CITED4 interacts via its conserved C-terminal region with the CH1 domain of CREBBP and EP300, and interacts with all TFAP2/AP-2 isoforms. It is also synonymous with “transcriptional coactivator 4,” “MSG-1 related protein 2,” or “MRG2.”
  • CITED4 represents a 184-a.a. human polypeptide having the following polypeptide sequence:
  • the CITED4 polypeptide is about 98% identical to its counterpart in Pan troglodytes (Chimpanzee; Access No. H2PYS8 or K7BVD1), including 100% identical in the 61 C-terminal residues; 96% identical to its counterpart in Macaca mulatta (Rhesus macaque; Access No. I2CVG1), including 100% identical in 60 of the 61 C-terminal residues; 87% identical to its counterpart in Bos taurus (Bovine; Access No. Q2HJ78), including 100% identical in 60 of the 61 C-terminal residues except for a conserved E to D change; 84% identical to its counterpart in Cavia porcellus (Guinea pig; Access No.
  • H0W249 including 100% identical in 59 of the 60 C-terminal residues; 77% identical to its counterpart in Mus musculus (Mouse; Access No. A2A7E7), including 100% identical in 49 of the 50 C-terminal residues; and 73% identical to its counterpart in Rattus norvegicus (Rat; Access No. Q99MA0), including 100% identical in 51 of the 53 C-terminal residues.
  • CITED4 represents a mammalian polypeptide at least about 75%, 80%, 85%, 90%, 95%, 98% identical to SEQ ID NO: 3, such as the above-referenced mammalian CITED4 polypeptides.
  • CITED4 represents a mammalian polypeptide that is a functional fragment of any of the above-referenced mammalian sequences, such as a functional fragment of SEQ ID NO: 3.
  • the functional fragment comprises or consists essentially of or consists of the C-terminal 48, 49, 50, 51, 52, 53, 54, 55, 60, 61, 62, or 63 residues of SEQ ID NO: 3 or its counterpart fragments in any of the mammalian sequences, such as those referenced above.
  • the invention also provides fusion proteins of any of the above functional fragments with a heterologous polypeptide, such as a epitope tag, or any heterologous protein that does not substantially negatively impact the function of the functional fragment.
  • a heterologous polypeptide such as a epitope tag
  • the functional fragment or fusion thereof stimulates cardiomyocyte growth, proliferation, and/or regeneration, to substantially the same extent (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95% or nearly 100% or more efficient/effective) as the wildtype CITED4 polypeptide.
  • the functional fragment or fusion thereof acts as transcriptional coactivator for Transcription Factor AP-2 (TFAP2)/AP-2; enhances estrogen-dependent transactivation mediated by estrogen receptors; functions as an inhibitor of transactivation by HIF1A by disrupting HIF1A interaction with CREB Binding Protein (CREBBP); and/or is involved in regulation of gene expression during development and differentiation of blood cells, endothelial cells and mammary epithelial cells, to substantially the same extent (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95% or nearly 100% or more efficient/effective) as the wildtype CITED4 polypeptide.
  • TFAP2 Transcription Factor AP-2
  • CREBBP CREB Binding Protein
  • CITED4 is encoded by a polynucleotide encoding any of the above-referenced mammalian CITED4 polypeptides or functional fragments or fusions thereof.
  • the human CITED4 polypeptide may be encoded by a polynucleotide represented by NCBI nucleotide RefSeq. NM_133467.2.
  • the polynucleotide may be an mRNA (including a synthetic, modified mRNA, such as one described herein), or a DNA.
  • the encoding polynucleotide may be within a suitable vector (such as an AAV vector) that is capable of directing the expression of CITED4 in a suitable mammalian host cell, such as a cardiomyocyte or precursor thereof.
  • the CITED4 polypeptide or functional fragments or fusions thereof, and the miR-222, a precursor (e.g., pre-miR-222), and mimics of the invention (or “the subject agents”) can be expressed in vitro and in vivo from a vector or expression construct.
  • a “vector” or “expression construct” is used to deliver a nucleic acid of interest (such as a nucleic acid encoding the subject agents) to the target expression site, such as the interior of a cell/cardiomyocytes.
  • a nucleic acid of interest such as a nucleic acid encoding the subject agents
  • target expression site such as the interior of a cell/cardiomyocytes.
  • vectors are known in the art, including (but not limited to) linear polynucleotides, polynucleotides associated with ionic or amphiphillic compounds, plasmids, and virus-based vectors.
  • the term “vector” includes an autonomously replicating plasmid or a virus-based vector.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, and the like.
  • a vector or expression construct can be replicated in a living cell, or it can be made
  • an expression vector for expressing a subject agent comprises a promoter operably linked to a polynucleotide encoding a sequence of the subject agent (e.g., CITED4 polypeptide or miR-222).
  • a promoter operably linked to a polynucleotide encoding a sequence of the subject agent (e.g., CITED4 polypeptide or miR-222).
  • “Operably linked” or “under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase (such as RNA Pol II or Pol III), and/or expression of the encoded CITED4.
  • the polynucleotide encoding miR-222 may encode the primary microRNA sequence, the precursor-microRNA sequence, the mature miRNA sequence, or the star (e.g. minor) sequence of miR-222.
  • the polynucleotide encoding miR-222 can be about 18 to about 2000 nucleotides in length, about 70 to about 200 nucleotides in length, about 20 to about 50 nucleotides in length, or about 18 to about 25 nucleotides in length.
  • the nucleic acid encoding a CITED4 polypeptide or a miR-222 is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced transcription machinery, required to initiate the specific transcription of a gene.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the polynucleotide sequence of interest.
  • CMV cytomegalovirus
  • a promoter By employing a promoter with well-known properties, the level and pattern of expression of a subject agent of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the subject agent.
  • exemplary regulatory elements that may be employed, in the context of the present invention, to regulate the expression of the subject agent of interest, may include (but are not limited to): viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the subject agent of interest in an expression construct.
  • any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the polynucleotide.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • Illustrative promoter and/or enhancer include: Ig heavy chain (Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.; 1990); Ig light chain (Queen et al., 1983; Picard et al., 1984); T-Cell Receptor (Luria et al., 1987; Winoto et al., 1989; Redondo et al., 1990); HLA DQ ⁇ and/or DQ ⁇ (Sullivan et al., 1987); ⁇ -Interferon (Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn et al., 1988); Interleukin-2 (Greene et al., 1989
  • Inducible Elements include: MT II promoter inducible by Phorbol Ester (TFA) or Heavy metals (Palmiter et al., 1982; Haslinger et al., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989); MMTV (mouse mammary tumor virus) promoter inducible by Glucocorticoids (Huang et al., 1981; Lee et al., 1981; Majors et al., 1983; Chandler et al., 1983; Ponta et al., 1985; Sakai et al., 1988); ⁇ -Interferon promoter inducible by poly(rI)x or poly(rc) (Tavernier et al., 1983); Adenovirus 5 E2 promoter inducible by E1A (I
  • the inducible promoter is a tetracyclin-inducible promoter, in a tetracycline-controlled transcriptional activation system/construct, which is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline or Dox, a more stable tetracycline analogue).
  • a tetracyclin-inducible promoter in a tetracycline-controlled transcriptional activation system/construct, which is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline or Dox, a more stable tetracycline analogue).
  • Tet-Off The two most commonly used inducible expression systems for research of eukaryote cell biology are Tet-Off and Tet-On.
  • the Tet-Off system activates expression in the absence of Dox (Bujard et al., Proc. Natl. Acad. Sci. U.S.A. 89 (12):5547-5551, 1992), whereas the Tet-On system activates expression in the presence of Dox.
  • the Tet-Off system employs the tetracycline transactivator (tTA) protein, which is a fusion protein of the E. coli TetR (tetracycline repressor) and the activation domain of HSV transcription factor VP16.
  • tTA tetracycline transactivator
  • the resulting tTA protein is able to bind to DNA at specific TetO operator sequences.
  • TetO operator sequences In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter such as the CMV promoter.
  • the entirety of several TetO sequences with a minimal promoter is known as tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter.
  • TRE tetracycline response element
  • TRE-controlled genes can be repressed by tetracycline and its derivatives such as Dox. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes.
  • Tet-On system works similar, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the tTA protein is capable of binding the operator only if bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred over Tet-Off for its faster responsiveness.
  • the Tet-On Advanced transactivator (also known as rtTA2 S -M2) is an alternative version of Tet-On that shows reduced basal expression, and functions at a 10-fold lower Dox concentration than Tet-On.
  • its expression is considered to be more stable in eukaryotic cells due to being human codon optimized and utilizing 3 minimal transcriptional activation domains. It was discovered as one of two improved mutants after random mutagenesis of the Tet Repressor part of the transactivator gene (Urlinger et al., Proc. Natl. Acad. Sci. U.S.A. 97 (14):7963-7968, 2000).
  • Tet-On 3G (also known as rtTA-V10) is similar to Tet-On Advanced because they were derived from the same predecessor. It is also human codon optimized and composed of 3 minimal VP16 activation domains. However, the Tet-On 3G protein has 5 amino acid differences compared to Tet-On Advanced which appear to increase its sensitivity to Dox even further. Tet-On 3G is sensitive to 100-fold less Dox than the original Tet-On (Zhou et al., Gene Ther. 13 (19):1382-1390, 2006).
  • Tet-Off and Tet-On expression systems can both be used in generating transgenic mice, which conditionally express gene of interest.
  • the Tet-On system (including Tet-On Advanced transactivator and Tet-On 3G) is used with the methods and constructs of the invention.
  • Tet-Off system is used with the methods and constructs of the invention.
  • muscle specific promoters and more particularly, cardiac specific promoters.
  • myosin light chain-2 promoter (Franz et al., 1994; Kelly et al., 1995), the alpha actin promoter (Moss et al., 1996), the troponin I promoter (Bhaysar et al., 1996); the Na + /Ca 2+ exchanger promoter (Barnes et al., 1997), the dystrophin promoter (Kimura et al., 1997), the alpha7 integrin promoter (Ziober and Kramer, 1996), the brain natriuretic peptide promoter (LaPointe et al., 1996) and the alpha B-crystallin/small heat shock protein promoter (Gopal-Srivastava, 1995), alpha myosin heavy chain promoter (Yamauchi-Takihara et al., 1989) and the ANF promoter (LaPointe et al.,
  • a polyadenylation signal may be included to effect proper polyadenylation of the gene transcript where desired.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator is also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct.
  • a marker in the expression construct.
  • Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • Immunologic markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with or as an indicator of the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • expression vectors may be introduced into cells.
  • Cardiovascular gene therapy is currently the third most popular application for gene therapy behind cancer and the collection of inherited monogenic diseases, and provides a new avenue for host of therapeutic uses including therapeutic angiogenesis, myocardial protection, regeneration and repair, prevention of restenosis following angioplasty, prevention of bypass graft failure and risk-factor management.
  • adenoviral vector 23.3% of all trials
  • retroviral vector about 20% of all trials
  • viruses have been less widely used and include vaccinia virus (7.9% of trials), poxvirus (5.0%), adeno-associated virus (4.9%), and herpes simplex virus (3.1%).
  • poxvirus and vaccinia virus adenovirus and retrovirus
  • adenovirus and vaccinia virus naked DNA and adenovirus
  • adenovirus and modified vaccinia Ankara virus naked DNA and vaccinia virus.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein.
  • the expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992).
  • adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • adenovirus type 5 of subgroup C may be used as starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention.
  • Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus E1 region.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, as described by Karlsson et al. (1986), or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993).
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as retrovirus, vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
  • the expression constructs In order to effect expression of the subject agents encoded by the expression constructs, the expression constructs must be delivered into a target cell, such as a cardiomyocyte. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • a target cell such as a cardiomyocyte.
  • This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • Synthetic vectors not based on viral systems can also be used in the methods of the invention.
  • the simplest nonviral gene delivery system uses “naked” DNA, which, when injected directly into certain tissues, particularly muscle, produces significant levels of gene expression, although lower than those achieved with viral vectors. Naked DNA has been used in about 18.3% of the clinical trial by 2012, and it is the most popular nonviral system used in clinical trials. Lipofection, the second most used non-viral delivery in clinical trials (used in 5.9% of all trials), involves cationic lipid/DNA complexes. A small number of trials have also used a range of modified bacteria (20 trials) or brewer's yeast strains (seven trials). These methods can also be used in the methods of the invention to deliver a subject agent or a coding nucleic acid.
  • non-viral methods for the transfer of expression constructs into cultured mammalian cells include naked DNA encoding a subject agent, calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979), and lipofection or lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988), and bacteria or yeast mediated delivery. Some of these techniques may be successfully adapted for in viv
  • the nucleic acid encoding the subject agents may be positioned and expressed at different sites.
  • the nucleic acid encoding the subject agents may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a polynucleotide of interest may also be transferred in a similar manner in vivo and express the gene product.
  • a naked DNA expression construct into cells may involve particle bombardment.
  • This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987).
  • Several devices for accelerating small particles have been developed.
  • One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment.
  • DNA encoding a particular polynucleotide of interest may be delivered via this method and still be incorporated by the present invention.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang and Wu, 1990
  • the polynucleotide may be administered in combination with a cationic lipid or neutral lipid, or a combination of cationic and anionic lipids that together result in a neutral charge (see e.g. WO 05/007196 and WO 05/026372, which are herein incorporated by reference in their entireties).
  • cationic lipids include, but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP.
  • lipid or liposomal formulations including nanoparticles and methods of administration include, but are not limited to, U.S. Patent Publications 2003/0203865, 2002/0150626, 2003/0032615, and 2004/0048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids.
  • Methods used for forming particles are also disclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated by reference for those aspects.
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
  • a subject agent e.g., a CITED4 polypeptide or functional fragment or fusion thereof, or miR-222 or a mimic thereof
  • administration of a subject agent results in the improvement of one or more symptoms of the cardiovascular disease or pathological condition (e.g., myocardial infarction, heart failure, or cardiac remodeling).
  • the one or more improved symptoms can be, for example, increased exercise capacity, increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output, increased cardiac index, lowered pulmonary artery pressures, decreased left ventricular end systolic and diastolic dimensions, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, and decreased disease related morbidity or mortality.
  • expression of a subject agent in the heart cells of a subject can reduce infarct size by decreasing the loss of heart cells (e.g., decreasing apoptosis in the infarct zone).
  • expression of a subject agent in the heart cells of a subject can reduce fibrosis in the infarct zone.
  • expression of a subject agent in the heart cells of a subject can stabilize cardiac function.
  • the subject polypeptide may be expressed using synthetic, modified RNA as described in US 2012-0046346 A1 and US 2014-0186432 A1 (incorporated herein by reference), which described in detail compositions, methods, and kits comprising synthetic, modified RNAs for changing the phenotype of a cell or cells, by expressing a desired polypeptide (e.g., CITED4 or functional fragments or fusions thereof) in a target cell or tissue or in vivo.
  • compositions, and kits do not utilize exogenous DNA or viral vector-based methods for the expression of protein(s), and thus, do not cause permanent modification of the genome or have the potential for unintended mutagenic effects.
  • synthetic, modified RNAs are included within the scope of the term “expression construct” as it is used in the instant application.
  • compositions, methods, and kits described herein are based upon the direct introduction of the synthetic, modified RNAs into a cell, which translated the RNA into a desired polypeptide.
  • the effect of the cellular innate immune response is mitigated by using synthetic RNAs that are modified in a manner that avoids or reduces the response. Avoidance or reduction of the innate immune response permit sustained expression from exogenously introduced RNA.
  • sustained expression of the subject polypeptide e.g., CITED4 or functional fragments or fusions thereof
  • the modified, synthetic RNAs can be introduced to a cell in order to induce exogenous expression of the subject polypeptide.
  • the ability to direct exogenous expression of the subject polypeptide using the modified, synthetic RNAs described herein is useful, for example, in the treatment of disease or disorders caused by an endogenous defect in a cell or organism that impairs or prevents the ability of that cell or organism to produce the subject polypeptide (e.g., a subject unable to produce, or unable to produce sufficient quantities of, the subject polypeptide, either as a result of genetic defect, or as a result of injury).
  • compositions and methods comprising the modified, synthetic RNAs described herein can be used for the purposes of gene therapy.
  • the synthetic, modified RNA molecule comprises at least two modified nucleosides.
  • the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′ deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-dimethylguanos
  • the synthetic, modified RNA molecule further comprises a 5′ cap.
  • the 5′ cap is a 5′ cap analog.
  • the 5′ cap analog is a 5′ diguanosine cap.
  • the synthetic, modified RNA molecule does not comprise a 5′ triphosphate.
  • the synthetic, modified RNA molecule further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof.
  • the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.
  • the synthetic, modified RNA molecule is further treated with an alkaline phosphatase.
  • the innate immune response comprises expression of a Type I or Type II interferon.
  • the innate immune response comprises expression of one or more IFN signature genes selected from the group consisting of IFN ⁇ , IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.
  • a cell such as a cardiomyocyte or precursor or progeny thereof contacted with a synthetic, modified RNA molecule encoding a subject polypeptide, where the synthetic, modified RNA molecule comprises one or more modifications, such that introducing the synthetic, modified RNA molecule to the cell results in a reduced innate immune response relative to the cell contacted with a synthetic RNA molecule encoding the polypeptide not comprising the one or more modifications.
  • the synthetic, modified RNA molecule contacted with the cell comprises at least two modified nucleosides.
  • the two modified nucleosides are selected from the group consisting of 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2′-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2′ fluorouridine, pseudouridine, 2′-O-methyluridine (Um), 2′ deoxy uridine (2′ dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2′-O-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), N6,N6,2′-O-trimethyladenosine (m62Am), 2′-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2′-O-methylguanosine (Gm), N2,7-d
  • the synthetic, modified RNA molecule contacted with the cell further comprises a 5′ cap.
  • the 5′ cap is a 5′ cap analog.
  • the 5′ cap analog is a 5′ diguanosine cap.
  • the synthetic, modified RNA molecule contacted with the cell does not comprise a 5′ triphosphate.
  • the synthetic, modified RNA molecule contacted with the cell further comprises a poly(A) tail, a Kozak sequence, a 3′ untranslated region, a 5′ untranslated region, or any combination thereof.
  • the poly(A) tail, the Kozak sequence, the 3′ untranslated region, the 5′ untranslated region, or the any combination thereof comprises one or more modified nucleosides.
  • the synthetic, modified RNA molecule contacted with the cell is further treated with an alkaline phosphatase.
  • the synthetic, modified RNA molecule comprises a coding region (e.g., a coding region for CITED4), said coding region having an altered G/C content as compared to the G/C content of the coding region comprising nucleotides 220-774 of SEQ ID NO: 275 of US 2014-0186432 A1.
  • the coding region which has an altered G/C content may be selected from the group of nucleic acid sequences consisting of SEQ ID NO: 1421, 1794, 2414, 3034, 3654, 4274, and 8408-8494 of US 2014-0186432 A1.
  • the G/C content in the coding region is decreased as compared to the G/C content in the coding region of SEQ ID NO: 275 of US 2014-0186432 A1.
  • the coding region with a decreased G/C content may be selected from the group consisting of SEQ ID NO: 2414, 3034, 3654, 4274, and 8408-8494 of of US 2014-0186432 A1.
  • the G/C content in the coding region is the same as compared to the G/C content in the coding region of SEQ ID NO: 275 of of US 2014-0186432 A1.
  • the coding region with the same G/C content may be SEQ ID NO: 1794 of of US 2014-0186432 A1.
  • the nucleic acid comprises at least one untranslated region 5′ relative to the coding region and at least one untranslated region 3′ relative to the coding region.
  • the 5′ untranslated region may be heterologous to the coding region of the nucleic acid, and/or the 3′ untranslated region may be heterologous to the coding region of the nucleic acid.
  • the 5′ untranslated region and the 3′ untranslated region are heterologous to the coding region of the nucleic acid.
  • the nucleic acid comprises at least two stop codons.
  • the nucleic acid encods SEQ ID NO: 890 of of US 2014-0186432 A1, wherein said nucleic acid comprises a coding region, said coding region having an altered G/C content as compared to the G/C content of the coding region comprising nucleotides 220-774 of SEQ ID NO: 275 of US 2014-0186432 A1.
  • the innate immune response comprises expression of a Type I or Type II interferon, and the expression of the Type I or Type II interferon is not increased more than three-fold compared to a reference from a cell which has not been contacted with the synthetic modified RNA molecule.
  • the innate immune response comprises expression of one or more IFN signature genes selected from the group consisting of IFN ⁇ , IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, and where the expression of the one of more IFN signature genes is not increased more than six-fold compared to a reference from a cell which has not been contacted with the synthetic modified RNA molecule.
  • IFN signature genes selected from the group consisting of IFN ⁇ , IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20, and where the expression of the one of more IFN signature genes is not increased more than six
  • the polypeptide encoded by the synthetic, modified RNA molecule introduced to the cell alters a function or a developmental phenotype of the cell.
  • the developmental phenotype is a developmental potential.
  • the developmental potential is decreased.
  • the developmental potential is increased.
  • the polypeptide encoded by the synthetic, modified RNA molecule introduced to the cell promotes the growth, proliferation, and/or regeneration of cardiomyocytes, or a precursor or progeny thereof.
  • the polypeptide encoded by the synthetic, modified RNA molecule is a transcription factor, or function as a transcription factor.
  • the cell is a human cell. In other embodiments, the cell is not a human cell. In certain embodiments, the cell is an adult cell. In certain embodiments, the cell is not aneonatal cell (e.g., neonatal carbiomyocyte).
  • the cell or its immediate precursor cell(s) has been subjected to at least 3 separate rounds of contacting with the exogenously introduced modified synthetic RNA encoding the subject polypeptide.
  • the cell has a reduced expression of a Type I or Type II IFN relative to a cell subjected to at least 3 separate rounds of contacting with an exogenously introduced non-modified, synthetic RNA encoding the subject polypeptide. In certain embodiments, the cell has a reduced expression of at least one IFN-signature gene relative to a cell subjected to at least 3 separate rounds of contacting with an exogenously introduced non-modified synthetic RNA encoding the subject polypeptide.
  • the IFN-signature gene is selected from the group consisting of IFN ⁇ , IFNB1, IFIT, OAS1, PKR, RIGI, CCL5, RAP1A, CXCL10, IFIT1, CXCL11, MX1, RP11-167P23.2, HERC5, GALR3, IFIT3, IFIT2, RSAD2, and CDC20.
  • composition comprising at least one modified, synthetic RNA encoding a subject polypeptide, and cell growth media.
  • contacting of the cell population or progeny cells thereof is performed in vitro, ex vivo, or in vivo.
  • aspects described herein provide methods of treating subjects in need of cellular therapies.
  • an effective amount of a population of any of the progenitor, multipotent, oligopotent, lineage-restricted, fully or partially differentiated cells, generated using any of the compositions or methods comprising synthetic, modified RNAs described herein, is administered to a subject in need of a cellular therapy.
  • a method of treating a subject in need of a cellular therapy comprising: administering to a subject in need of a cellular therapy an effective amount of a population of cells (e.g., cardiomyocytes) produced by contacting a cell population or progeny cells thereof with at least one synthetic, modified RNA encoding a subject polypeptide (preferably for at least three consecutive times).
  • a population of cells e.g., cardiomyocytes
  • the method further comprises a step of obtaining an autologous cell from the subject and generating a population of cardiomyocytes from the autologous cell by contacting the cell population or progeny cells thereof with at least one synthetic, modified RNA encoding a subject polypeptide (preferably for at least three consecutive times).
  • kits comprising: a) a container with at least one synthetic, modified RNA molecule comprising at least two modified nucleosides and encoding a subject polypeptide, and b) packaging and instructions therefor.
  • the kit further comprises a container with cell culture medium.
  • the kit further comprises an IFN inhibitor.
  • the kit further comprises valproic acid.
  • the kit further comprises a non-implantable delivery device or an implantable delivery device to deliver the at least one synthetic, modified RNA.
  • the non-implantable delivery device is a pen device.
  • the implantable delivery device is a pump, semi-permanent stent, or reservoir.
  • the invention provides methods of using a subject miR-222 microRNA or a precursor (e.g., pre-miR-222) or mimic thereof, or a CITED4 polypeptide or functional fragments or fusions thereof, for treating various cardiovascular diseases or pathological conditions described herein.
  • the cardiovascular disease or pathological condition is myocardial infarction or ischemic injury; adverse remodeling after ischemic injury or infarction; myocarditis; heart failure (congestive); cardiomyopathies, such as ischemic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy, tachycardia-mediated cardiomyopathy, stress-induced (takotsubo) cardiomyopathy, amyloid cardiomyopathy, arrhythmogenic right ventricular dysplasia, or unclassified cardiomyopathies, for example left ventricular noncompaction or endocardial fibroelastosis; or valvular heart disease, such as aortic stenosis, aortic regurgitation, mitral stenosis, mitral regurgitation, mitral prolapse, pulmonary stenosis, pulmonary regurgitation, tricuspid stenosis, or tricuspid regurgitation.
  • cardiomyopathies such as ischemic cardiomyopathy,
  • the cardiovascular disease or pathological condition is ischemic injury, such as one due to myocardial infarction, and/or fibrosis resulting therefrom.
  • the cardiovascular disease or pathological condition is in an individual currently having, have had in the past, or is at risk of having such cardiovascular disease or pathological condition.
  • the individual also referred to as a subject, may be a mammalian species, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent (including an experimental animal such as a mouse or rat), or primate, such as a human or a non-human primate.
  • Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), zoo animals (e.g., lions, giraffes, etc.), but are not so limited.
  • Preferred subjects are human subjects (individuals).
  • the human subject may be a pediatric, adult or a geriatric subject.
  • an “adult” patient include any individual that is not considered a newborn or neonatal individual (e.g., a human at least 1 year-old, 2-year old, 3-year old, 5-year old, 10-year old, 15-year old, 20-year old or older). In certain embodiments, however, an “adult” patient refers to a human at least about 15-year old, or at least about 18-year old.
  • the present invention contemplates the treatment and prevention of, among other things, post-MI remodeling of cardiac tissues that surround an infarct as well as the subsequent development of heart failure in a subject.
  • Treatment regimens would vary depending on the clinical situation, with earliest intervention being sought. However, long-term maintenance for at least some period of time post-MI would appear to be appropriate in most circumstances. It also may be desirable to treat with the subject agent intermittently, or to vary which of the subject agent is given, in order to maximize the protective effects.
  • one or more of the subject agent can be used in combination with other therapeutic modalities, such as those more “standard” pharmaceutical cardiac therapies.
  • other therapies include, without limitation, so-called “ ⁇ -blockers,” mineralocorticoid antagonists, anti-hypertensives, cardiotonics, antithrombotics, vasodilators, hormone antagonists, iontropes, diuretics, endothelin receptor antagonists, calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors, angiotensin type 2 antagonists and cytokine blockers/inhibitors, and/or HDAC inhibitors.
  • Combinations may be achieved by contacting cardiac cells with a single composition or a pharmacological formulation that includes one or more of the subject agents and a second cardiac therapy, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes one or more of the subject agents and the other includes the second cardiac therapy.
  • administration of one or more of the subject agents may precede or follow administration of the other cardiac agent(s) by intervals ranging from minutes to weeks.
  • the other cardiac agent and the subject agents are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the cardiac agent and the subject agents would still be able to exert an advantageously combined effect on the cell.
  • Pharmacological therapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the Physicians Desk Reference , Klaassen's The Pharmacological Basis of Therapeutics, Remington's Pharmaceutical Sciences , and The Merck Index , Eleventh Edition, incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration to any individual patient will, in any event, determine the appropriate dose for that individual subject, and such individual determinations are within the skill of those of ordinary skill in the art.
  • Non-limiting examples of a pharmacological therapeutic agent that may be used in combination with the miRNA modulators of the present invention include an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, an antibacterial agent or a combination thereof.
  • administration of an agent that lowers the concentration of one of more blood lipids and/or lipoproteins may be combined with a cardiovascular therapy according to the present invention, particularly in treatment of atherosclerosis and thickenings or blockages of vascular tissues.
  • an antihyperlipoproteinemic agent may comprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof.
  • Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate.
  • Non-limiting examples of resins/bile acid sequesterants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide.
  • Non-limiting examples of HMG CoA reductase inhibitors include lovastatin (mevacor), pravastatin (pravochol) or simvastatin (zocor).
  • Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.
  • Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine.
  • miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, ⁇ -benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8,11,14,17-eicosapentaenoic acid, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, ⁇ -oryzanol, pantethine, pentaerythritol tetraacetate, ⁇ -phenylbutyramide, pirozadil, probucol (lorelco), ⁇ -sitosterol, sultosilic acid-piperazine salt, tiadenol, triparanol and xenbucin.
  • a non-limiting example of an antiarteriosclerotic includes pyridino
  • administering may be combined with administration of a miRNA modulator, particularly in treatment of atherosclerosis and vasculature (e.g., arterial) blockages.
  • a miRNA modulator particularly in treatment of atherosclerosis and vasculature (e.g., arterial) blockages.
  • antithrombotic and/or fibrinolytic agents include anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof.
  • antithrombotic agents that can be administered orally, such as, for example, aspirin and warfarin (Coumadin).
  • the subject agent can be combined with one or more anticoagulants.
  • anticoagulants include acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol and warfarin.
  • the subject agent can also be combined with an antiplatelet agent and/or a thromobolytic agent.
  • antiplatelet agents include aspirin, a dextran, dipyridamole (persantin), heparin, sulfinpyranone (anturane) and ticlopidine (ticlid).
  • thrombolytic agents include tissue plaminogen activator (activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), anistreplase/APSAC (eminase).
  • an agent that may enhance blood coagulation may be used in combination with a miRNA modulator.
  • blood coagulation promoting agents include thrombolytic agent antagonists and anticoagulant antagonists.
  • anticoagulant antagonists include protamine and vitamin K1.
  • Non-limiting examples of thrombolytic agent antagonists that can be combined with a miRNA modulator include amiocaproic acid (amicar) and tranexamic acid (amstat).
  • Non-limiting examples of antithrombotics include anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.
  • a subject agent can be combined with an antiarrhythmic agent for the treatment of cardiovascular conditions.
  • antiarrhythmic agents include Class I antiarrhythmic agents (sodium channel blockers), Class II antiarrhythmic agents (beta-adrenergic blockers), Class III antiarrhythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrhythmic agents.
  • Sodium channel blockers include, but are not limited to, Class IA, Class IB and Class IC antiarrhythmic agents.
  • Class IA antiarrhythmic agents include disppyramide (norpace), procainamide (pronestyl) and quinidine (quinidex).
  • Class IB antiarrhythmic agents include lidocaine (xylocalne), tocamide (tonocard) and mexiletine (mexitil).
  • Class IC antiarrhythmic agents include encamide (enkaid) and flecamide (tambocor).
  • beta blockers otherwise known as a ⁇ -adrenergic blockers, ⁇ -adrenergic antagonists or Class II antiarrhythmic agents, include acebutolol (sectral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, labetalol, levobun
  • the beta blocker comprises an aryloxypropanolamine derivative.
  • aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolo
  • Class III antiarrhythmic agents include agents that prolong repolarization, such as amiodarone (cordarone) and sotalol ( ⁇ -pace).
  • Class IV antiarrythmic agents also known as calcium channel blockers, include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a miscellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexyline.
  • arylalkylamine
  • miscellaneous antiarrhythmic agents that can also be combined with a subject agent include, but are not limited to, adenosine (adenocard), digoxin (lanoxin), acecamide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecamide, ipatropium bromide, lidocaine, lorajmine, lorcamide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.
  • adenosine adenocard
  • digoxin lanoxin
  • acecamide acecamide
  • ajmaline amoproxan
  • aprindine bre
  • the subject agent can be administered in combination with an antihypertensive agent.
  • antihypertensive agents include sympatholytic, alpha/beta blockers, alpha blockers, anti-angiotensin II agents, beta blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives.
  • an alpha blocker also known as an ⁇ -adrenergic blocker or an ⁇ -adrenergic antagonist
  • an alpha blocker include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine.
  • an alpha blocker may comprise a quinazoline derivative.
  • Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin.
  • an antihypertensive agent is both an alpha and beta adrenergic antagonist.
  • Non-limiting examples of an alpha/beta blocker comprise labetalol (normodyne, trandate).
  • Non-limiting examples of anti-angiotensin II agents include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists.
  • Non-limiting examples of angiotensin converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril.
  • angiotensin II receptor blocker also known as an angiotensin II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS)
  • angiocandesartan eprosartan, irbesartan, losartan and valsartan.
  • Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherally acting sympatholytic.
  • Non-limiting examples of a centrally acting sympatholytic also known as an central nervous system (CNS) sympatholytic, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet).
  • Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a ⁇ -adrenergic blocking agent or a alphal-adrenergic blocking agent.
  • Non-limiting examples of a ganglion blocking agent include mecamylamine (inversine) and trimethaphan (arfonad).
  • Non-limiting examples of an adrenergic neuron blocking agent include guanethidine (ismelin) and reserpine (serpasil).
  • Non-limiting examples of a ⁇ -adrenergic blocker include acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol (naimodyne, trandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren).
  • Non-limiting examples of alphal-adrenergic blocker include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin).
  • a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator) that can be co-administered with a miRNA modulator of the invention.
  • a vasodilator comprises a coronary vasodilator.
  • Non-limiting examples of a coronary vasodilator include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis( ⁇ -diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexyline, pimethylline, trapidil, tricromyl, trimetazidine,
  • a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator.
  • a chronic therapy vasodilator include hydralazine (apresoline) and minoxidil (loniten).
  • a hypertensive emergency vasodilator include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil.
  • miscellaneous antihypertensives include ajmaline, ⁇ -aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil.
  • an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a N-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reserpine derivative or a suflonamide derivative.
  • arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol.
  • Non-limiting examples of benzothiadiazine derivatives include althizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachlomethiazide and trichlormethiazide.
  • Non-limiting examples of N-carboxyalkyl (peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril.
  • Non-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine.
  • Non-limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.
  • Non-limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine.
  • Non-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine.
  • Non-limiting examples of quanternary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate.
  • Non-limiting examples of reserpine derivatives include bietaserpine, deserpidine, rescinnamine, reserpine and syrosingopine.
  • Non-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.
  • a subject agent can be co-administered with a vasopressor.
  • Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure.
  • Non-limiting examples of a vasopressor also known as an antihypotensive, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine.
  • a subject agent can be administered in combination with a treatment for congestive heart failure.
  • agents for the treatment of congestive heart failure include, but are not limited to, anti-angiotensin II agents, afterload-preload reduction treatment, diuretics and inotropic agents.
  • Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide), an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride
  • an animal patient that can not tolerate an angiotensin antagonist may be treated with a combination therapy, such as administration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate) with a subject agent.
  • a combination therapy such as administration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate) with a subject agent.
  • a subject agent can also be combined with an inotropic agent.
  • the inotropic agent is a positive inotropic agent.
  • a positive inotropic agent also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, aminone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain
  • an inotropic agent is a cardiac glycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor.
  • a cardiac glycoside includes digoxin (lanoxin) and digitoxin (crystodigin).
  • Non-limiting examples of a ⁇ -adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol.
  • Antianginal agents may comprise organonitrates, calcium channel blockers, beta blockers and combinations thereof.
  • Non-limiting examples of organonitrates also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole).
  • a subject agent is co-administered with endothelin for treatment of a cardiovascular disease.
  • Endothelin is a 21-amino acid peptide that has potent physiologic and pathophysiologic effects that appear to be involved in the development of heart failure.
  • the effects of ET are mediated through interaction with two classes of cell surface receptors.
  • the type A receptor (ET-A) is associated with vasoconstriction and cell growth while the type B receptor (ET-B) is associated with endothelial-cell mediated vasodilation and with the release of other neurohormones, such as aldosterone.
  • Pharmacologic agents that can inhibit either the production of ET or its ability to stimulate relevant cells are known in the art.
  • Inhibiting the production of ET involves the use of agents that block an enzyme termed endothelin-converting enzyme that is involved in the processing of the active peptide from its precursor inhibiting the ability of ET to stimulate cells involves the use of agents that block the interaction of ET with its receptors.
  • endothelin receptor antagonists include Bosentan, Enrasentan, Ambrisentan, Darusentan, Tezosentan, Atrasentan, Avosentan, Clazosentan, Edonentan, sitaxsentan, TBC 3711, BQ 123, and BQ 788.
  • the secondary therapeutic agent that can be combined with the subject agent may comprise a surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery.
  • Surgery, and in particular a curative surgery may be used in conjunction with other therapies, such as the subject agent of the invention and one or more other agents.
  • Such surgical therapeutic agents for vascular and cardiovascular diseases and disorders are well known to those of skill in the art, and may comprise, but are not limited to, performing surgery on an organism, providing a cardiovascular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable cardioverter defibrillator to the subject, mechanical circulatory support or a combination thereof.
  • a mechanical circulatory support that may be used in the present invention comprise an intra-aortic balloon counterpulsation, left ventricular assist device or combination thereof.
  • compositions comprising a subject agent will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. Colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the subject agents.
  • Colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the subject agents.
  • fat emulsions that are suitable for delivering the nucleic acids of the invention to tissues, such as cardiac muscle tissue, include INTRALIPID®, LIPOSYN®, LIPOSYN® II, LIPOSYN® III, Nutrilipid, and other similar lipid emulsions.
  • An exemplary colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos.
  • Aqueous compositions of the present invention comprise an effective amount of the agent, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically acceptable or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or nucleic acids of the compositions.
  • the active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Pharmaceutical compositions comprising the subject agents may also be administered by catheter systems or systems that isolate coronary circulation for delivering therapeutic agents to the heart. Various catheter systems for delivering therapeutic agents to the heart and coronary vasculature are known in the art. Some non-limiting examples of catheter-based delivery methods or coronary isolation methods suitable for use in the present invention are disclosed in U.S. Pat. Nos.
  • compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • these preparations are sterile and fluid to the extent that easy injectability exists.
  • Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
  • the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the present invention generally may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups of the protein can also be
  • solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • aqueous solution for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
  • a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards.
  • an effective amount also referred to as a therapeutically effective amount, of a subject agent or pharmaceutical composition thereof, is an amount sufficient to ameliorate at least one symptom associated with the cardiovascular disease or pathological condition.
  • the therapeutically effective amount to be included in pharmaceutical compositions may depend, in each case, upon several factors, e.g., the type, size and condition of the patient to be treated, the intended mode of administration, the capacity of the patient to incorporate the intended dosage form, etc. One of ordinary skill in the art would be able to determine empirically an appropriate therapeutically effective amount.
  • the formulations of the present invention for human use comprise the agent, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof or deleterious to the inhibitory function of the active agent.
  • the formulations should not include oxidizing agents and other substances with which the agents are known to be incompatible.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • All methods include the step of bringing into association the agent with the carrier, which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the agent with the carrier(s) and then, if necessary, dividing the product into unit dosages thereof.
  • Formulations suitable for parenteral administration conveniently comprise sterile aqueous preparations of the agents, which are preferably isotonic with the blood of the recipient.
  • suitable such carrier solutions include phosphate buffered saline, saline, water, lactated ringers or dextrose (5% in water).
  • Such formulations may be conveniently prepared by admixing the agent with water to produce a solution or suspension, which is filled into a sterile container and sealed against bacterial contamination.
  • sterile materials are used under aseptic manufacturing conditions to avoid the need for terminal sterilization.
  • Such formulations may optionally contain one or more additional ingredients among which may be mentioned preservatives, such as methyl hydroxybenzoate, chlorocresol, metacresol, phenol and benzalkonium chloride.
  • preservatives such as methyl hydroxybenzoate, chlorocresol, metacresol, phenol and benzalkonium chloride.
  • Buffers may also be included to provide a suitable pH value for the formulation. Suitable such materials include sodium phosphate and acetate. Sodium chloride or glycerin may be used to render a formulation isotonic with the blood. If desired, the formulation may be filled into the containers under an inert atmosphere such as nitrogen or may contain an anti-oxidant, and are conveniently presented in unit dose or multi-dose form, for example, in a sealed ampoule.
  • the administration of the pharmaceutical composition or formulation to a patient can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection.
  • the administration may be by continuous infusion, or by single or multiple boluses.
  • the dosage may vary depending upon such factors as the patient's age, weight, gender, general medical condition, and previous medical history.
  • delivery to the heart of a pharmaceutical formulation described herein comprises coronary artery infusion.
  • coronary artery infusion involves inserting a catheter through the femoral artery and passing the catheter through the aorta to the beginning of the coronary artery.
  • targeted delivery of a therapeutic to the heart involves using antibody-protamine fusion proteins, such as those previously describe (Song E. et al., Nature Biotechnology, 23(6):709-717, 2005).
  • CITED4 is increased in exercised hearts, and demonstrated that CITED4 drives proliferation of neonatal cardiomyocytes in vitro (Bostrom et al., Cell, 143:1072-1083, 2010, see FIG. 1 ).
  • Applicant has assessed the ability of CITED4 expression to induce proliferation in adult cardiomyocytes.
  • forced expression of CITED4 in adult cardiomyocytes resulted in comparable levels of CITED4 expression to that seen in neonatal cardiomyocytes.
  • Applicant was unable to demonstrate markers of cell proliferation in >500 adult cardiomyocytes examined in vitro.
  • Example 1 Applicant tested the ability of CITED4 to induce cardiomyocyte proliferation in vivo, by using regulated, controllable expression of CITED4 in a bigenic transgenic system.
  • cDNA encoding the full length mouse CITED4 polypeptide was inserted into a cardiomyocyte-specific expression vector which utilizes an attenuated myosin heavy chain promoter (as previously described in Sanbe et al., “Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter,” Circ. Res. 92 (6):609-616, 2003, incorporated herein by reference), from which the expression of the mature full length CITED4 polypeptide is controlled through expression of a tetracyclin-regulated transcription factor (cf above reference).
  • Transgenic mouse harboring the expression construct was created according to oocyte injection using standard protocols.
  • Expression of the CITED4 polypeptide can be induced by withdrawing tetracycline or doxycycline from the drinking water or food of the transgenic animal, or can be turned off by restoring the tetracycline or doxycycline-containing water or chow. There is virtually no exogenous CITED4 expression in the presence of tetracycline or doxycycline in the chow or drinking water. After inducing expression of the exogenous CITED4, adult cardiomyocyte proliferation can be measured by checking the expression level of biomarkers (such as EdU, phospho-histone H3, ki67, and/or Aurora B kinase in cardiomyocytes identified by virtue of Troponin T expression).
  • biomarkers such as EdU, phospho-histone H3, ki67, and/or Aurora B kinase in cardiomyocytes identified by virtue of Troponin T expression.
  • CITED4 expression was induced in hearts subjected to ischemia-reperfusion injury, similar to that seen in patients who suffer a myocardial infarction and then receive first line reperfusion therapy.
  • the initial reduction (24 hr) in cardiac function was similar in both CITED4 expressing and control hearts.
  • the CITED4-expressing hearts showed substantial recovery of cardiac function and reduction in fibrosis (scar) at 5 weeks after injury.
  • conditional expression of CITED4 is achieved using a transgenic construct expressing CITED4 under the control of a Tet-inducible promoter (see Example 2 above and Sanbe et al., supra). Stained histological sections comparing heart tissues from CITED4-expresing animal to those from negative control show substantial recovery of damaged cardiac tissues and reduced scar/fibrosis.
  • mice To identify microRNA that could be central regulators of physiological cardiac growth, Applicant subjected mice to either voluntary wheel running or a ramp swimming exercise model (Taniike et al., Circulation 117: 545-552, 2008) for three weeks in comparison to sedentary controls. As shown in FIGS. 9A-9E , both models induced mild cardiac hypertrophy, and microRNAs were profiled in hearts samples from each exercise model in comparison to sedentary controls.
  • the TaqMan rodent miRNAarray (A+B set v3.0), which includes 641 unique assays specific to mouse, was used to investigate microRNAs involved in the cardiac exercise response. Five cardiac samples were used from mice exercised in each of the models for three weeks in comparison to matched sedentary controls.
  • miR-222 emerged as a particularly interesting candidate that was increased 2.1- and 2.8-fold in the swimming and running exercise models, respectively (p ⁇ 0.003 and 0.02).
  • miR-222 was one of two microRNAs (along with miR-191) that increased cardiomyocyte size in vitro, and one of four (along with miR-139, -27a and -484) that increased EdU incorporation, a measure of proliferation ( FIGS. 10A and 10B ).
  • FIGS. 10A and 10B Subsequent studies showed that only miR-222, miR-339, and miR-486 induced a physiological pattern of myosin heavy chain isoform expression ( FIG. 10C ). Based on the unique convergence of these characteristics, miR-222 was further studied below.
  • miR-222 is a highly conserved micro-RNA that has been reported by others to increase in serum of exercised athletes. The role of miR-222 in the heart, however, is unknown.
  • miR-222 is necessary for the short-term growth of the heart and cardiomyocytes in response to exercise. miR-222 also appears to contribute to the increase in markers of proliferation seen in cardiomyocytes after exercise.
  • miR-222 is a highly conserved member of a microRNA cluster encoded on the X chromosome, which also includes miR-221 (Felli et al., Proc Natl Acad Sci USA 102: 18081-18086, 2005; Galardi et al., J Biol Chem 282: 23716-23724, 2007). Its function in the heart is unknown.
  • miR-222 induced an increase in cardiomyocyte size ( FIG. 11A ).
  • miR-222 expression caused an increase in markers of cardiomyocyte proliferation (as seen in the initial characterization) as well as cell number ( FIG. 11C ).
  • miR-222 induced a pattern of gene expression in cardiomyocytes consistent with healthy or physiological growth, including an increase in myosin heavy chain (MHC)- ⁇ / ⁇ ratio, as well as decreases in expression of ANP, BNP, and ⁇ -skeletal actin mRNAs ( FIG. 11E ).
  • MHC myosin heavy chain
  • miR-222 has also been reported to increase in the plasma of athletes in response to both acute and chronic exercise (Baggish et al., Dynamic Regulation of Circulating MicroRNA during Acute Exhaustive Exercise and Sustained Aerobic Exercise Training. J Physiol (Lond), 2011), suggesting the potential human relevance of these observations.
  • miR-221 expression in hearts nor miR-222 expression in skeletal muscle was increased in our exercised mice (data not shown).
  • Tg-miR-222 inducible (doxycycline-off) cardiac-specific miR-222 transgenics (Tg-miR-222) to investigate the effects of cardiac miR-222 expression.
  • Tg-miR-222 mice manifest regulated, cardiac-specific miR-222 expression that was ⁇ 6.5-fold increased 4 weeks after removal of doxycycline from chow ( FIG. 12A ).
  • Tg-miR-222 mice appeared grossly normal at baseline after induction of cardiac miR-222 expression with normal heart size ( FIG. 12B ) and cardiac function (as measured by ECG analysis).
  • miR-222 is necessary for growth of the heart in response to exercise, miR-222 expression, even at levels higher than those induced by exercise, appears to be insufficient to recapitulate the exercised-heart phenotype.
  • Tg-miR-222 mice were withdrawn from doxycycline and subsequently subjected to ischemia-reperfusion injury (RI) induced by 30 minutes coronary ligation and reperfusion.
  • RI ischemia-reperfusion injury
  • Tg-miR-222 mice showed no difference in initial infarct size or degree of cardiac dysfunction in comparison to controls ( FIGS. 13A & 13B ).
  • Tg-miR-222 mice did not, but instead maintained both chamber dimension and cardiac function ( FIG. 13B ).
  • miR-222-expressing mice had substantially better cardiac function ( FIG. 13B ), as well as an almost 70% reduction in cardiac fibrosis ( FIG. 13C ).
  • in vivo miR-222 expression after ischemic injury was associated with an increase in EdU incorporation in cardiomyocytes but a decrease in EdU incorporation non-cardiomyocytes, which are predominantly fibroblasts ( FIG. 13D ).
  • miR-222 expression preserved cardiac structure and function, enhanced functional recovery (as measured by fractional shortening), and is sufficient to mitigate adverse remodeling after ischemic injury. Meanwhile, miR-222 expression reduced scar formation at 6 weeks in comparison to control mice ( FIG. 6 ) (fibrosis measured by Masson Trichrome Staining), after ischemic injury.
  • Applicant used a combination of bioinformatic analyses with expression profiling in miR-222-expressing cardiomyocytes to identify putative target candidates for miR-222, e.g., by transcript profiling on cardiomyocytes expressing miR-222 in comparison to control vector.
  • GSE59641 Gene Expression Omnibus
  • a combination of expression profiling results with miR-222 targets predicted by two bioinformatic programs (Targetscan, Pictar) ( FIG. 12A ) identified four relevant potential targets whose expression decreased in cardiomyocytes with miR-222 expression. These include the cell cycle inhibitor, p27(kip1), the protein kinases HIPK-1 and -2, and a transcriptional repressor, Hmbox1 ( FIG. 7 ).
  • HIPK1, HIPK2, and Homeobox1 have not previously been linked to miR-222 and have no known roles in the heart.
  • miR-222 expression in cardiomyocytes in vitro was sufficient to reduce expression of all four putative targets (p27, HIPK1, HIPK2, Homeobox-1) from 35 to 55 percent, while a specific miR-222 inhibitor increased their expression ( FIG. 14A ). These changes in RNA levels were paralleled by changes in protein expression for each of the targets ( FIG. 14B ).
  • HIPK1, HIPK2, and Homeobox-1 were direct targets of miR-222.
  • miR-222 expression had no effect on luciferase activity of the control reporter without a miR-222 binding site (data not shown).
  • miR-222 expression induced a reduction in luciferase activity for each of the wild-type 3′UTR constructs but importantly, but had no significant effect when the miR-222 binding sites were mutated (data not shown), demonstrating that the binding interaction is required for this effect.
  • HIPK1, HIPK2 and Homeobox-1 mRNAs are all direct targets of miR-222, as has been shown for p27.
  • these targets have distinct contributions to the phenotypic effects of miR-222, as shown in FIGS. 8A-8B .
  • siRNA knockdown of either p27 or HIPK1 was sufficient to induce an increase in EdU incorporation in cardiomyocytes ( FIG. 14D ) as well as an increase in cardiomyocyte cell number ( FIG. 14E ).
  • Homeobox-1 knockdown did not affect cell number but caused an increase in cell size ( FIG. 14F ).
  • p27 knockdown actually caused a decrease in cell size, perhaps reflecting active cell division; HIPK2 had no significant effect in either assay.
  • miR-222 has been reported to increase in the plasma of healthy young athletes response to exercise, and exercise has beneficial effects in heart failure patients, Applicant sought to determine whether these observations could be related.
  • changes in circulating miR-222 in twenty-eight heart failure patients were examined after acute cardiopulmonary exercise using a bicycle ergometer. Baseline patient characteristics are shown in Table 1, and included patients with stable chronic heart failure (NYHA Class II-IV) with both preserved and reduced systolic function. Exercise duration ranged from 2.5 to 11 minutes on a standardized protocol (Myers, International journal of sports medicine 26 Suppl 1: S49-55, 2005).
  • This example demonstrates that miR-222 expression can be increased in vivo in a tissue specific manner using an AAV vector.
  • an AAV-9 based viral expression vector encoding miR-222 (AAV9-miR-222) was injected venously into mice (about 10 11 viral particles in 100 ⁇ L).
  • AAV9-miR-222 the same vector encoding a GFP reporter was similarly injected to a control mice group.
  • expression of miR-222, and miR-221 as control were measured in heart and liver of the injected mice.
  • mice injected by the AAV9-GFP reporter had no discernible difference in miR-221 and miR-222 expression, either in the heart or in the liver.
  • mice injected by the AAV9-miR222 construct had dramatic increase of miR-222 expression in the heart, while the heart expression level of miR-221 was essentially the same as that in the AAV9-GFP mice.
  • specific viral vectors such as AAV9 can be used to deliver and express miR-222 or other nucleic acid of the invention in a specific tissue (e.g., heart).
  • TAQMAN® Rodent miRNA microarray cards and assays TAQMAN® Rodent miRNA microarray cards and assays, TAQMAN® MicroRNA Reverse Transcription Kit, TAQMAN® Universal Master Mix II, no UNG, AMBION® PRE-MIRTM miRNA Precursors, SIPORTTM NEOFXTM Transfection Agent, OPTI-MEM® I Reduced Serum Media, High-Capacity cDNA Reverse Transcription Kit, Power SYBR® Green PCR Master Mix, Alexa Fluo (488 and 594) conjugated antibodies, CLICK-IT® EdU ALEXA FLUOR® 488 Imaging Kits, PROLONG® Gold Antifade Reagent with DAPI were purchased from Life technologies.
  • Antibodies against Ki-67, TNNT2, P27, GAPDG, and nonimmune IgG were from Abcam.
  • Antibodies to ⁇ -actinin and HMBOX-1 were from Sigma and Bioworld.
  • HRP-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.
  • In vivo LNATM microRNA inhibitors were purchased from Exiqon, and MICRONTM miRNA agomir was obtained from Ribobio.
  • siRNAs for p27(kip1), Hmbox1, Hipk1, and Hipk2 and negative controls were purchased from Invitrogen.
  • microRNA precursors (PRE-MIRTM miRNA Precursors) and negative controls were ordered from Invitrogen.
  • Transfection of siRNAs (20 ⁇ M), LNA-modified antimiR oligonucleotides (20 ⁇ M), or microRNA precursors (0.4 ⁇ M) were carried out using Lipofectamine RNAiMAX (Invitrogen) as recommended by the manufacturer. Unless otherwise indicated, a multiplicity of infection (MOI) of 20 was used for adenoviral transfection.
  • MOI multiplicity of infection
  • RNA Isolation RNA Isolation, Quantitative Real Time PCR (qRT-PCR) and Microarray
  • RNA from cultured cells and tissues was isolated Tryzol (Invitrogen) following the manufacturers' instructions.
  • qRT-PCR for microRNA was performed on cDNA generated from 100 ng of total RNA using the TaqMan microRNA protocol (Invitrogen).
  • qRT-PCR for mRNA was performed on cDNA generated from 200 ng of total RNA using the high capacity cDNA reverse transcription kit protocol (Invitrogen).
  • Amplification and detection of specific products were performed on a Biorad CFX384 qPCR System.
  • U6 or sno202 was used as an internal control for microRNA template normalization and GADPH or U6 was used for mRNA template normalization.
  • Relative gene expression was calculated by comparing cycle times (Ct) for each target PCR as previously described (Liu et al., 2009).
  • Ct cycle times
  • RNA was isolated from 400 ⁇ L plasma using the mirVana PARIS isolation kit (Ambion, Austin, Tex.) according to the manufacturer's instructions for plasma samples without enrichment for small RNAs.
  • the Caenorhabditis elegans miRNA cel-miR-39 was added at 50 pmol/L as a control after adding equal volumes of denaturing solution.
  • the Bulge-LoopTM miRNA qPCR Primer Set (RiboBio) was used to detect miR-222 expression by qRT-PCRs with SYBR Green PCR Master Mix (Bio-Rad, Hercules, Calif., USA) using the 7900HT Fast Real-Time PCR System with 10 ⁇ L of PCR master mix containing 1 ⁇ L of Forward primer, 1 ⁇ L of Reverse primer, 5 ⁇ L SYBR Green (2 ⁇ ), 2 ⁇ L of RT products, and 1 ⁇ L of ddH 2 O. Cycling parameters were as follows: denaturation at 95° C. for 15 s; annealing at 60° C. for 30 s; and elongation at 72° C.
  • NRVMs were plated in a 6 cm BD Primaria tissue culture dish. Twenty-four hours after plating, cells were transfected with 20 ⁇ M LNA, 0.4 ⁇ M RNA oligo precursor, or 20 ⁇ M siRNA, using lipofectamine RNAiMax overnight. Cells were then synchronized in serum-free media for 24 hours and subsequently cultured in low serum media. Forty-eight hours after transfection, cells were labeled with 20 ⁇ M EdU for 24 hours. Before harvesting, cells were incubated with 50 ⁇ M Mitotracker orange for 45 minutes. Collected cells were stained by using the protocol of Click-iT EdU Flow Cytometry Assay (Invitrogen). Stained cells were analyzed in a 5-laser LSR II machine in BIDMC flow core facility. At least 10,000 events were recorded by flow cytometry in each treatment. Flowjo7.6.1 was used to analyze flow data.
  • protein was isolated from cultured NRVMs and hearts, and equal amounts of protein as determined by BCA protein assay kit (Pierce) were subjected to SDS-PAGE. After membrane transfer, immunoblotting was conducted using primary antibodies to p27 (Kip1) (1:1000, Cell Signaling #3698), HIPK1 (1:500, Abcam ab90103), HIPK2 (1:500, Cell Signaling #5091), and HMBOX1 (1:500, Abcam ab101140). HSP90 (1:1000) was used as a loading control.
  • cardiac troponin-I Abcam ab56357
  • cardiac troponin-T Abcam ab10214
  • ⁇ -actinin Sarcomeric, Sigma A7732
  • Ki67 Cell Signaling #9129
  • phospho-Histone H3 Cell Signaling, #3377
  • a reporter plasmid was constructed by inserting a fragment of the 3′-UTR of Hipk1, Hipk2 or Hmbox1 mRNA containing the putative miR-222 binding site into a firefly luciferase reporter plasmid psiCHECK-2 (Promega).
  • 3′-UTRs with mutated miR-222 binding sites for Hipk1, Hipk2 or Hmbox1 mRNA were generated using the Quikchange Site-directed Mutagenesis kit (Agilent).
  • COS7 cells were co-transfected with the wild-type or mutated reporter constructs (100 ng) and Ambion pre-miR miR-222 precursor or scrambled control (1 ⁇ M) using Lipofectamine 2000 (Invitrogen). 48 hours after transfection, cells were lysed, and relative luciferase expression was measured using a SpectraMax M5 plate reader using a dual luciferase reporter system (Promega).
  • mice Male C57BL6/J mice swam in water tanks by using a protocol as described (Taniike et al., 2008). In brief, mice began with two 10-minute swimming sessions separated by at least 4 hrs. Sessions were increased by 10 minutes each day until reaching 90 minute sessions, twice a day. The protocol was stopped after 21 days. During swimming, mice were supervised at all times. Twenty-four hours after the last swimming session, exercised mice were sacrificed and tissues were collected.
  • mice Male C57BL6/J mice aged 10-12 weeks were subjected to voluntary cage wheel exercise as previously described (Bourajjaj et al., 2008). Briefly, mice were individually housed in a cage equipped with an 11.5-cm-diameter running wheel with a 5.0-cm-wide running surface equipped with a digital magnetic counter activated by wheel rotation. Daily values of exercise time and running distance were recorded for each exercised animal throughout the exercise period. After three weeks of exercise, mice were sacrificed and tissues harvested.
  • a tetracycline-off binary ⁇ -MHC transgene system was used as previously described (Sanbe et al., 2003).
  • a 388 bp fragment containing mmu-miR-222 was amplified from mouse genomic DNA and confirmed by sequencing. The fragment was then cloned into a vector, and a large fragment released by digestion with Not I was microinjected into FVB oocytes and transferred to pseudopregnant mice. Cardiac-specific, doxycycline-regulated miR-222 expression was confirmed in line 12, which was used for all the experiments presented.
  • doxycycline was administered in the food using a special diet formulated by Purina (625 mg/kg in pellets). To induce miR-222 expression, mice at 10 to 12 weeks old were fed normal chow (without doxycycline for 4 weeks).
  • LNA-antimiR injections were performed as described (Grueter et al., 2012). Briefly, 12-week-old C57Bl6 male mice were injected subcutaneously or via tail vein with 10 mg/kg of locked nucleic acid (LNA)-modified antimiR-222 (LNA-antimiR-222) or scrambled control (LNA-SC) reconstituted in saline. Both LNA-antimiR oligonucleotides were purchased from Exiqon.
  • LNA locked nucleic acid
  • LNA-antimiR-222 locked nucleic acid-modified antimiR-222
  • LNA-SC scrambled control
  • LNA-antimiR-222 The sequence of LNA-antimiR-222 is +g*t*+a*+g*c*+c*a*+g*+a*t*+g*+t*a*g*+c, in which the “+” and “*” signs indicated an LNA residue and the modified phosphorothioate linkages respectively.
  • the mice were injected for 3 consecutive days and then given a weekly maintenance injection throughout the experiments.
  • Transgenic FVB or C57BL/6 wild type mice were subjected to ischemia-reperfusion as previously described (Matsui et al., 2002). Briefly, left anterior descending artery (LAD) was ligated with 7-0 silk. Five minutes into ischemia, 50 ⁇ l of fluorescent microspheres (10 ⁇ m FluoSpheres, Molecular Probes) were injected into the LV cavity. Following 30 min LAD occlusion, the LAD ligature was released, and reperfusion was visually confirmed. After the indicated time interval of reperfusion, mice were sacrificed and tissues collected for analyses.
  • LAD left anterior descending artery
  • the area-at-risk and myocardial infarct size 24 hours after reperfusion were determined by fluorescent microscopy (for FluoSpheres) and staining with 2,3,5-triphenyltetrazolium chloride (TTC) respectively, as reported previously (Matsui et al., 2002).
  • EdU 50 mg/kg was administered subcutaneously every other day for two weeks to identify new DNA synthesis. Sham-operated mice served as controls. All surgeries and analyses were performed by investigators blinded to treatment group and/or genotype.
  • Echocardiography was performed on conscious mice by using a GE Vivid7 with i13L probe (14 MHZ) as described previously (Das et al., 2012). Briefly, parasternal long-axis views, short-axis views and 2-D guided M-mode images of short axis at the papillary muscle level were recorded. Echocardiography data were analyzed by investigators blinded to treatment group and genotype. The average of at least three measurements was used for every data point from each mouse.
  • RNA isolation and PCR were performed as described below.

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Publication number Priority date Publication date Assignee Title
WO2007062827A1 (fr) 2005-11-30 2007-06-07 Deutsches Krebsforschungszentrum, Stiftung Des Öffentlichen Rechts Cited4 en tant que marqueur de pronostic dans des tumeurs oligodendrogliales

Family Cites Families (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0273085A1 (fr) 1986-12-29 1988-07-06 IntraCel Corporation Procédé pour faire entrer des acides nucléiques dans des cellules eucaryotes
US6867195B1 (en) 1989-03-21 2005-03-15 Vical Incorporated Lipid-mediated polynucleotide administration to reduce likelihood of subject's becoming infected
AU637800B2 (en) 1989-08-31 1993-06-10 City Of Hope Chimeric dna-rna catalytic sequences
JP3257675B2 (ja) 1990-10-12 2002-02-18 マックス−プランク−ゲゼルシャフト ツール フェルデルング デル ビッセンシャフテン エー．ファウ． 修飾リボザイム
DE4216134A1 (de) 1991-06-20 1992-12-24 Europ Lab Molekularbiolog Synthetische katalytische oligonukleotidstrukturen
US5652094A (en) 1992-01-31 1997-07-29 University Of Montreal Nucleozymes
US5981505A (en) 1993-01-26 1999-11-09 The Trustees Of The University Of Pennsylvania Compositions and methods for delivery of genetic material
WO1995003843A1 (fr) 1993-07-30 1995-02-09 The Regents Of The University Of California Catheter de perfusion endocardiaque
US5972901A (en) 1994-03-23 1999-10-26 Case Western Reserve University Serpin enzyme complex receptor--mediated gene transfer
JP4285766B2 (ja) 1994-03-23 2009-06-24 オハイオ ユニバーシティ 緻密にした核酸および細胞へのデリバリ
US5844107A (en) 1994-03-23 1998-12-01 Case Western Reserve University Compacted nucleic acids and their delivery to cells
US6077835A (en) 1994-03-23 2000-06-20 Case Western Reserve University Delivery of compacted nucleic acid to cells
US5837533A (en) 1994-09-28 1998-11-17 American Home Products Corporation Complexes comprising a nucleic acid bound to a cationic polyamine having an endosome disruption agent
US5840710A (en) 1994-12-09 1998-11-24 Genzyme Corporation Cationic amphiphiles containing ester or ether-linked lipophilic groups for intracellular delivery of therapeutic molecules
US5716824A (en) 1995-04-20 1998-02-10 Ribozyme Pharmaceuticals, Inc. 2'-O-alkylthioalkyl and 2-C-alkylthioalkyl-containing enzymatic nucleic acids (ribozymes)
DE69637256T2 (de) 1996-01-16 2008-06-19 Sirna Therapeutics, Inc., Boulder Synthese von Methoxynukleoside und enzymatische Nukleisäure Moleküle
US6416510B1 (en) 1997-03-13 2002-07-09 Biocardia, Inc. Drug delivery catheters that attach to tissue and methods for their use
US6217900B1 (en) 1997-04-30 2001-04-17 American Home Products Corporation Vesicular complexes and methods of making and using the same
DE69834038D1 (de) 1997-07-01 2006-05-18 Isis Pharmaceutical Inc Zusammensetzungen und verfahren zur verabreichung von oligonukleotiden über die speiseröhre
US6749617B1 (en) 1997-11-04 2004-06-15 Scimed Life Systems, Inc. Catheter and implants for the delivery of therapeutic agents to tissues
ATE340560T1 (de) 1999-05-24 2006-10-15 Introgen Therapeutics Inc Verfahren und zusammensetzungen für nichtvirale gentherapie zur behandlung von hyperproliferativen krankheiten
US20020042388A1 (en) 2001-05-01 2002-04-11 Cooper Mark J. Lyophilizable and enhanced compacted nucleic acids
WO2002032396A2 (fr) 2000-10-16 2002-04-25 Massachusetts Institute Of Technology Particules a base de lipides-proteines-glucides pour transport d'acides nucleiques
JP2004535388A (ja) 2001-04-30 2004-11-25 ターゲティッド ジェネティクス コーポレイション 脂質含有薬物送達複合体およびそれらの生成方法
WO2003093449A2 (fr) 2002-05-06 2003-11-13 Nucleonics, Inc. Procedes d'administration d'acides nucleiques
ES2559828T3 (es) 2003-07-16 2016-02-16 Protiva Biotherapeutics Inc. ARN de interferencia encapsulado en lípidos
NZ592917A (en) 2003-09-15 2012-12-21 Protiva Biotherapeutics Inc Stable polyethyleneglycol (PEG) dialkyloxypropyl (DAA) lipid conjugates
AU2005216564A1 (en) 2004-02-26 2005-09-09 Osprey Medical Inc. Isolating cardiac circulation
US20070203445A1 (en) 2004-02-26 2007-08-30 V-Kardia Pty Ltd Isolating cardiac circulation
US7722596B2 (en) 2004-02-26 2010-05-25 Osprey Medical, Inc. Regional cardiac tissue treatment
WO2006089340A2 (fr) 2005-02-23 2006-08-31 V-Kardia Pty Ltd Administration d'un polynucleotide a un tissu cardiaque
US9072765B2 (en) 2009-05-20 2015-07-07 Board Of Regents, The University Of Texas System Identification of micro-RNAs involved in post-myocardial infarction remodeling and heart failure
CA2796464C (fr) 2010-04-16 2021-08-03 Immune Disease Institute, Inc. Expression de polypeptide prolongee a partir d'arn synthetiques modifies et utilisations de celle-ci
AU2013243948A1 (en) 2012-04-02 2014-10-30 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with human disease

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007062827A1 (fr) 2005-11-30 2007-06-07 Deutsches Krebsforschungszentrum, Stiftung Des Öffentlichen Rechts Cited4 en tant que marqueur de pronostic dans des tumeurs oligodendrogliales

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
Bostrom et al. (2010) Cell, vol. 143, 1072-1083. *
Boström et al., "C/EBPb Controls Exercise-Induced Cardiac Growth and Protects against Pathological Cardiac Remodeling," Cell, 143:1072-1083 (2010).
Caroli et al., "Potential therapeutic role of microRNAs in ischemic heart disease," J. Cardiol. 61:315-320 (2013).
Chistiakov et al., "Strategies to deliver microRNAs as potential therapeutics in the treatment of cardiovascular pathology," Drug Deliv., 19(8):392-405 (2012).
Ding et al., "MicroRNAs in cardiac hypertrophy: angels or devils," Wiley Interdisciplinary Reviews: RNA, 2(1):124-134 (2010).
Felli et al., "MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation," Proc. Natl. Acad. Sci. USA, 102(50):18081-18086 (2005).
Huang and Jia, "Construction of HCC-targeting artificial miRNAs using natural miRNA precursors," Exper. Thera. Med., 6:209-215 (2013).
Liu et al., "Cell-specific effects of miR-221/222 in vessels: Molecular mechanism and therapeutic application," J. Molec. Cell. Cardiol., 52:245-255 (2012).
Liu et al., "miR-222 Is Necessary for Exercise-Induced Cardiac Growth and Protects Against Pathological Cardiac Remodeling," Abstract 238, American Heart Association's Basic Cardiovascular Sciences 2014 Scientific Sessions: "Pathways to Cardiovascular Therapeutics," 2014; see also Circulation Research, 115(1)Suppl 1:229 (2014).
Liu et al., "miR-222 Is Necessary for Exercise-Induced Cardiac Growth and Protects against Pathological Cardiac Remodeling," Cell Metabolism, 21:584-595 (2015).
Liu et al., EMBASE Caesar accession No. 1153, "MIR-222 is necessary for excercise-induced cardiac growth and protects against pathological cardiac remodeling," 4 pages (2015).
Nagai et al. (2012) Am J. Heart Circ. Physiol., vol. 303, H501-H512. *
Ryall et al., "Phenotypic screen quantifying differential regulation of cardiac myocyte hypertrophy identifies CITED4 regulation of myocyte elongation," J. Molec. Cell. Cardiol., 72:74-84 (2014).
Wolfram et al. (2013) J Am Heart Assoc., vol. 2, e000119, pp. 1-11. *
Wolfram et al. (2013) J. Am. Heart Assoc., vol. 2, e000119, DOI: 10.1161/JAHA.113.000119, pp. 1-11. *
Wszolek et al., "A MicroRNA expression profile defining the invasive bladder tumor phenotype," Urologic Oncology: Seminars and Original Investigations, 29:794-801 (2011).
Zhang et al., "Dysregulation of angiogenesis-related microRNAs in endothelial progenitor cells from patients with coronary artery disease," Biochem. Biophys. Res. Comm., 405:42-46 (2011).
Zhang et al., "MicroRNA Deregulation in Right Ventricular Outflow Tract Myocardium in Nonsyndromic Tet ralogy of Fallot," Canadian J. Cardiol., 29:1695-1703 (2013).

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